Page 1 of 210 APPENDIX A

EVALUATION OF ARTICLES ON ATRAZINE AND ATRAZINE METABOLITES RELATED TO MAMMALIAN TOXICITY STUDIES SUGGESTING POTENTIAL ADVERSE HUMAN HEALTH OUTCOMESPage 2 of 210 A.1 CONTENT OF THIS APPENDIX

................................ ................................ ................................ .......................... 8

A.2 EFFECTS ON FEMA LES

................................ ................................ ................................ ................................ ... 10

A.2.1 C ASAS , E., B ONILLA , E., D UCOLOMB Y, B ETANCOURT M. 2010. D IFFERENTIAL EFFECTS OF HERBICIDES ATRAZI NE AND

FENOXAPROP

- ETHYL , AND INSECTICIDES DIA ZINON AND MALATHION , ON VIABILITY AND MAT URATION OF PORCINE O OCYTES IN VITRO

. T OXICOLO GY IN VITRO 24 (2010); 224

- 230.

................................ ................................ ................................ ................... 12 A.2.2 C ODER , P.S. 2010. A N O RAL D EVELOPMENTAL T OXICITY S TUDY OF A TRAZINE IN THE R AT , I NCLUDING C ROSS

- F OSTERING AND P AIR

- F EEDING

. U NPUBLISHED S YNGENTA STUDY

................................ ................................ ................................ ................ 13 A.2.3 C ODER , P.S. 2010. A N O RAL (G AVAGE ) S TUDY TO A SSESS THE P OTENTIAL FOR D ETECTING THE E STROGEN

- I NDUCED L UTEINIZING H ORMONE S URGE IN F EMALE R ATS

. S YNGENTA R EPORT

. ................................ ................................ .................. 15 A.2.4 C ODER P. (2011 E ). A TRAZINE : A N O RAL (G AVAGE ) S TUDY OF THE E FFECTS OF A TRAZINE ON TH E E STROUS C YCLE AND THE E STROGEN

- I NDUCED L UTEINIZING H ORMONE S URGE IN F EMALE R ATS

. F INAL R EPORT

. R EPLACES MRID 48180103) U NPUBLISHED

DATA FROM S YNGENTA C ROP P ROTECTION , I NC

. S TUDY COMPLETION DATE : F EBRUARY 4, 2011. R EPORT N UMBER : WIL

- 639060. MRID 4838100 1

................................ ................................ ................................ ................................ ...................... 17 A.2.5 C OOPER , R., B UCKALEW , A., F RAITES , M., G OLDMAN , J., L AWS , S., N AROTSKY , M., S TOKER , T. 2010. E VALUATING THE E FFECT OF THE C HLOROTRIAZINE H ERBICIDE A TRAZINE ON THE A MPLITUDE OF THE P RE

- OVULATORY LH SURGE IN THE L ONG

- E VANS R AT

. EPA I NTERNAL R EPORT

. ................................ ................................ ................................ ................................ ........ 21 A.2.6 D OOLEY , G.P., R EARDON , K.F., P RENNI , J.E., T JALKENS , R.B., L EGARE , M.E., F ORADORI , C.D., T ESSARI , J.E., H ANNEMAN , W.H. (2008). P ROTEOMIC ANALYSIS OF DIAMINOCHLOROTRIAZIN E ADDUCTS IN W ISTAR RAT PITUITARY GLANDS AND L Β T2 RAT PITUITARY CELLS

. C HEM R ES T OXICOL 21:844

- 851.

................................ ................................ ............................ 22 A.2.7 F RAITES , M.J.P., C OOPER , R.L., B UCKALEW , A., J AYARAMAN , S., M ILLS , L., AND L AWS , S.C. (2009). C HAR ACTERIZATION

OF THE H YPOTHALAMIC

- P ITUITARY

- A DRENAL A XIS R ESPONSE TO A TRAZINE AND M ETABOLITES IN THE F EMALE R AT

. T OX

. S CI

. 112(1):88

- 99.

................................ ................................ ................................ ................................ .......................... 24 A.2.8 G OJMERAC , T., P LEADIN , J., Z URIC , M., R AJKOVIC

- J ANJE , R., AND K ORSIC , M., 2004. S ERUM L UTEINIZING H ORMONE R ESPONSE TO A DMINISTRATION OF G ONADOTROPIN

- R ELEASING H ORMONE TO A TRAZINE

- T REATED G ILTS

. V ET H UMAN T OXICOL

. 46(5): 245

- 247.

................................ ................................ ................................ ................................ ........................ 27 A.2.9 J ULIANI , C., S ILVA

- Z ACARIN , E., S ANTOS , D. AND B OER , P. 2008. E FFECTS OF A TRAZINE ON F EMALE W ISTAR R ATS : M ORPHOLOGICAL A LTERATIONS IN O VARIAN F OLLICLES AND I MMUNOCYTOCHEMICAL L ABELING OF 90 K D A H EAT S HOCK P ROTEIN

. M ICRON

. 39:607

- 616.

................................ ................................ ................................ ................................ ............... 28 A.2.10 L AWS , S., F ARRELL , J., S TOKER , T. AND C OOPER , R. (2003) P UBERTAL D EVELOPMENT IN F EMALE W ISTAR R ATS

FOLLOWING E XPOSURE TO P ROPAZINE AND A TRAZINE B IOTRANSFORMATION B Y

- P RODUCTS , D IAMINO

- S

- C HLOROTRIAZINE AND H YDROXYATRAZINE

. T OXICOLOGICAL S CIENCES 76:190

- 200

. ................................ ................................ .............................. 30 A.2.11 M C M ULLIN , T.S., A NDERSEN , M.E., N AGAHARA , A. L UND , T.D., P AK , T., H ANDA , R.J., H ANNEMAN , W.H. (2004).
E VIDENCE THAT AT RAZINE AND DIAMINOCH LOROTRIAZINE INHIBIT THE ESTROGEN / PROGESTERONE INDUCED SURGE OF LUTEINIZING

HORMONE IN FEMALE S PRAGUE D AWLEY RATS WITHOUT C HANGING ESTROGEN REC EPTOR ACTION

. T OX

. S CI

. 79:278

- 286.

........... 31 A.2.12 S HIBAYAMA , H. K OTERA , T., S HINODA , Y., H ANADA , T. K AJIHARA , T., U EDA , M., T AMURA , H., I SHIBASHI , S., Y MASHITA , Y., AND O CHI , S. 2009. C OLLABORATIVE W ORK ON E VALUATION OF O VARI AN T OXICITY T WO

- OR F OUR

- WEEK R EPEATED D OSE S TUDIES AND F ERTILITY S TUDY OF A TRAZINE IN F EMALE R ATS

. ................................ ................................ .................... 32 A.2.13 S TOKER , T.E., Z ORRIL LA , L., S TRADER , L., G IBSON , E., K AMEL , A., Q IAN , Y., P ODHORNIAK , L. 2010. T HE D ISTRIBUTION OF A TRAZINE IN THE W ISTAR R AT F OLLOWING G ESTATIONAL /L ACTATIONAL E XPOSURES

. EPA I NTERNAL R EPORT

. ............................ 34 A.2.14 R AYNER , J.L., E NOCH , R.R., AND F ENTON , S.E. (2005) A DVERSE EFFECTS OF PR ENATAL EXPOSURE TO A TRAZINE DURING A

CRITICAL PERIOD OF M AMMARY GLAND GROWTH

. T OX

. S CI

. 87(1):255

- 266.

................................ ................................ ........ 36 A.2.15 D AVIS LK, M URR AS, B EST DS, F RAITES MJP, Z ORRILLA LM, M AROTSKY MG, S TOKER TE, G OLDMAN JM, C OOPER RL. 2011. T HE E FFECTS OF P RENATAL E XPOSURE TO A TRAZINE ON P UBERTAL AND P OSTNATAL R EPRODUCTIVE I NDICES IN THE F EMALE R AT

. R EPROD

. T OXICOL ., DOI :10.1016/ J

. REPROTOX .2011.04.004

................................ ................................ ................... 39 A.2.16 G OLDMAN J, D AVIS L, M URR A, C OOPER R. (2011). T HE I NFLUENCE OF 1, 2, AND 4 D AYS OF A TRAZINE T REATMENTS ON

THE LH S URGE IN O VARIECTOMIZED /E STRADIOL

- P RIMED R ATS : P RELIMINARY R EPORT TO THE O FFICE OF C HEM ICAL S AFETY AND P OLLUTION P REVENTION

. I NTERNAL R EPORT USEPA

................................ ................................ ................................ ......... 42 A.2.17 C ODER , P. S. (2011) A N O RAL (G AVAGE AND D IETARY ) S TUDY OF THE E FFECTS OF A TRAZINE ON THE S PONTANEOUS L UTEINIZING H ORMONE S URGE IN F EMALE S PRAGUE

- D AWLEY R ATS , WIL R ESEARCH L ABORATORIES , LLC, 1407 G EORGE R OAD , A SHLAND , OH. U NPUBLISHED

. S TUDY N UMBER : WIL

- 639080

................................ ................................ ........................... 43Page 3 of 210 A.2.18 C ODER , P. S. (2011) A N S TUDY OF THE E FFECTS OF S HORT

- T ERM A TRAZINE E XPOSURE ON THE E STROGEN

- I NDUCED L UTEINIZING H ORMONE (LH) S URGE IN O VARIECTOMIZED Y OUNG

- A DULT S PRAGUE D AWLEY F E MALE R ATS , WIL R ESEARCH L ABORATORIES , LLC, 1407 G EORGE R OAD , A SHLAND , OH. U NPUBLISHED

. S TUDY N UMBER : WIL

- 639091

............................... 47 A.2.19 C ODER , P. (2011 D ) A TRAZINE : A N O RAL (G AVAGE AND D IETARY ) S TUDY OF THE E FFECTS OF A TRAZINE ON F ERTILITY AND R EPRODUCTIVE P ERFORMANCE IN F EMALE L ONG E VANS AND S PRAGUE

- D AWLEY R ATS : F INAL R EPORT

. R EPORT N UMBER : WIL/639090, T ASK N O

. TK0052068. U NPUBLISHED STUDY

. P ERFORM ING L ABORATORY : WIL R ESEARCH L ABORATORIES , I NC

. 966

P

. C OMPLETION D ATE : A PRIL 6, 2011. MRID 48470001.

................................ ................................ ................................ 49

A.3 REPRODUCTIVE EF FECTS IN MALES

................................ ................................ ................................ ............... 51

A.3.1 A BARIKWU S., A DESLYAN A., O YELOJA , T., F AROMBI , E. 2010. C HANGES IN S PERM C HARACTERISTICS AND I NDUCTION OF O XIDATIVE S TRESS IN THE T ESTIS AND E PIDIDYMIS OF E XPERIMENTAL R ATS BY A H ERBICIDE , A TRAZINE

. A RCH

. E NVIRON C ONTAM T OXICOL 58:874

- 882. DOI 10.1007/ S 00244

- 009

- 9371

- 2.

................................ ................................ .......................... 56 A.3.2 B ETANCOURT , M., R ESÉNDIZ
, A., C ASAS , E. AND F IERRO , R. (2006) E FFECT OF TWO INSECTI CIDES AND TWO HERBIC IDES ON

THE PORCINE SPERM MO TILITY PATTERNS USIN G COMPUTER

- ASSISTED SEMEN ANALY SIS (CASA) IN VITRO

. R EPRO

. T OX

. 22:508

- 512.

................................ ................................ ................................ ................................ ................................ ................ 58 A.3.3 F RIEDMANN , AS. 2002. A TRAZINE INHIBITION O F TESTOSTERONE PRODU CTION IN RAT MALES F OLLOWING PERIPUBERTA L

EXP OSURE

. R EPRODUCTIVE T OXICOLOGY 16: 275

- 279.

................................ ................................ ................................ ..... 59 A.3.4 H ASE Y, T ATSUNO M, N ISHI T, K ATAOKA K, K ABE Y, Y AMAGUCHI Y, O ZAWA N, N ATORI M, H ANDA H AND W ATANABE H. (2008) A TRAZINE BINDS TO F1F0

- ATP SYNTHASE AND INHIBIT S MICROCHONDRIAL FUN CTION IN SPERM

. B IOCHEM B IOPHY R ESEARCH C OMM 366: 66

- 72.

................................ ................................ ................................ ................................ .................... 60 A.3.5 K ANDORI , H, S UZUKI S, A SAMOTO M, ET AL

. 2005. I NFLUENCE OF ATRAZINE ADMINISTRATION AND R EDUCTION OF

CALORIE INTAKE ON PR OSTATE CARCINOGENESI S IN PROBASIN /SV40 T ANTIGEN TRANSGENIC R ATS

. C ANCER S CI : 96: 221

– 226.

. 61 A.3.6 L AWS , S.C., H OTCHKISS , M., F ERRELL , J., J AYARAMAN , S., M ILLS , L., M ODIC , W., T INFO , N., F RAITES , M., S TOKER , T.,

AND C OOPER , R. (2009). C HLOROTRIAZINE H ERBICIDES AND M ETABOLITES A CTIVATE AN ACTH

- DEPENDENT R ELEASE OF C ORTICOSTERONE IN M ALE W ISTAR R ATS

. T OX

. S CI

. 112(1):78

- 87.

................................ ................................ ................... 63 A.3.7 L AWS , SC, S TOKER , TE, F ERRELL , JM, H OTCHKISS , MG, AND C OOPER RL. 2007. E FFECTS OF A LTERED F OOD I NTAKE D URING P UBERTAL D EVELOPMENT IN M ALE AND F EMALE W ISTAR R ATS

. T OXICOL

. S CI

. 100(1): 194

- 202.

................................ 64 A.3.8 M ARAVILLA

- G ALVAN , R., F IERRO , R., G ONZÁLES

- M ARQUEZ , H. G ÓMEZ

- A RROYO , S., J IMÉNEZ
, I R AND B ETANCOURT , M. 2009. E FFECTS OF A TRAZINE AND F ENOXAPROP

- E THYL ON C APACITATION AND THE A CROSOMAL R EACTION IN B OAR S PERM

. I NTERNATIONAL J OURNAL OF T OXICOLOGY

. V OLUME 2, N UMBER 1. PP

. 24

- 32.

................................ ................................ ..... 65 A.3.9 M U , H.
, S HANG , P. AND X U , J. (2008). T ESTICULAR T OXICITY AND M ECHANISMS OF C HLOROTOLURON C OMPOUNDS IN

THE M OUSE

. T OXICOLOGY M ECHANISMS AND M ETHODS

. 18: 399

- 403.

................................ ................................ .............. 67 A.3.10 P OGRMIC K, F A S, D AKIC V, K AISAREVIC S, AND K OVACEVIC R. 2009. A TRAZINE O RAL E XPOSURE OF P ERIPUBERTAL M ALE R ATS D OWNREGULATES S TEROIDOGENESIS G ENE E XPRESSION IN L EYDIG C ELLS

. T OXICOLOGICAL S CIENCES 111(1):189

- 197

. ........ 68 A.3.11 R AYNER J L., E NOCH R R, ET AL

. 2007. A TRAZINE

- INDUCED REPRODUCTIVE TRACT ALTERATIONS AF TER TRANSPLACENTAL

AND / OR LACT ATIONAL EXPOSURE IN MALE L ONG

- E VANS RATS

. T OX A PPL P HARMACOL : 218: 238

– 248.

................................ . 69 A.3.12 R OSENBERG , B.G., C HEN , H., F OLMER , J., L IU , J., P APADOPOULOS , V., AND Z IRKIN , B.R. (2008). G ESTATIONAL E XPOSURE

TO A TRAZINE : E FFECTS ON THE P OSTNATAL D EVELOPMENT OF M ALE O FFSPRING

. J. A NDROLOGY 29(3):304

- 311.

..................... 71 A.3.13 S TOKER TE, G UIDICI DL, L AWS SC, C OOPER RL. 2002. T HE E FFECTS OF A TRAZINE M ETABOLITES ON P UBERTY AND T HYROID F UNCTION IN THE M ALE W ISTAR R AT

. T OXICOLOGICAL S CIENCES 67: 198

- 206.

................................ ................................ ..... 73 A.3.14 S TOKER , T.E., L AWS , S.C., G UIDICI , D., AND C OOPER , R.L. 2000. T HE EFFECTS OF ATRAZI NE ON PUBERTY IN MAL E WISTAR

RATS : A N EVALUATION OF A PR OTOCOL FOR THE ASSESSMENT OF PU BERTAL DEVELOPMENT A ND THYROID FUNCTION

. T OXICOLOGICAL S CIENCES

. 58: 50

- 59.

................................ ................................ ................................ ................................ ................. 74 A.3.15 T RENTACOSTE , S V.. F RIEDMANN , AS, Y OUKER , RT, B RECKENRIDGE , CB, AND Z IRKIN , BR. 2001. A TRAZINE E FFECTS ON T ESTOSTERONE L EVELS AND A NDROGEN

- D EPENDENT R EPRODUCTIVE O RGANS IN P ERIPUBERTAL M ALE R ATS

. J OURNAL OF A NDROLOGY , V OL

. 22, N O

. 1, J ANUARY /F EBRUARY 2001 ; PP

. 142

- 148.

................................ ................................ ............ 75 A.3.16 V ICTOR

- C OSTA , AB, B ANDEIRA SMC, O LIVEIRA AG, M AHECHA GAB, O LIVEIRA , CA. 2010. C HANGES IN T ESTICULAR M ORPHOLOGY AND S TEROI DOGENESIS IN A DULT R ATS E XPOSED TO A TRAZINE

. R EPRODUCTIVE T OXICOLOGY

. OI 10.1016/ J

. REPROTOX .2009.12.006.

................................ ................................ ................................ ......................... 76 A.3.17 F RAITES MJP, N A ROTSKY MG, B EST DS, S TOKER TE, D AVIS LK, G OLDMAN JM, H OTCHKISS MG, K LINEFELTER GR, K AMEL A, Q IAN Y, P ODHORNIAK L, C OOPER RL. (2011). G ESTATIONAL A TRAZINE E XPOSURE E FFECTS ON M ALE R EPRODUCTIVE D EVELOPMENT AND M ETABOLITE D ISTRIBUTION IN THE D AM , F ETUS , AND N EONATE

. R EPROD

. T OXICOL ., DOI :10.1016/ J

. REPROTOX .2011.04.003

................................ ................................ ................................ ....................... 79Page 4 of 210 A.4 AROMATASE

................................ ................................ ................................ ................................ ................. 80
Summary: Effects on Aromatase and Steroidogenesis, Andrology

................................ ................................ .. 80 A.4.1 A LBANITO L, L APPANO R, M ADEO A, C HIMENTO A, P ROSSNITZ ER, C APPELLO AR, D OLCE V, A BONANTE S, P EZZI V, M AGGIOLINI M. (2008). G

- PROTEIN

- COUPLED RECEPTOR 30 AND ESTROGEN RECEPTO R

- Α ARE INVOLVED IN TH E PROLIFERATIVE

EFFECTS IND UCED BY ATRAZINE IN OVARIAN CANCER CELLS

. E NVIRON H EALTH P ERSPECT

. 116(12):1648

- 55.

.............................. 84 A.4.2 B ENACHOUR N, M OSLEMI S, S IPAHUTAR H, S ERALINI GE. (2007). C YTOTOXIC EFFECTS OF AROMATASE INHIBITION BY

XENOBIOTIC ENDOCRINE DISRUPTERS ALONE AND IN COMBINATION

. T OXICOLOGY AND A PPLIED P HARMACOLOGY

. 222:129

- 140.

. 86 A.4.3 F AN W, Y ANASE T, M ORINAGA H, G ONDA S, O KABE T, N OMURA M, H AYES T, T AKAYANAGI R, N AWATA H. (2007 A ).
H ERBICIDE ATRAZINE AC TIVATES SF

- 1 BY DIRECT AFFINITY A ND CONCOMITANT CO

- ACTIVATORS RECRUITME NTS TO IN DUCE AROMATASE

EXPRESSION VIA PROMO TER II. B IOCHEMICAL AND B IOPHYSICAL R ESEARCH C OMMUNICATIONS

. 355:1012

- 1018.

...................... 87 A.4.4 F AN W, Y AN ASE T, M ORINAGA H, G ONDO S, O KABE T, N OMURA M, K OMATSU T, M OROHASHI K, H AYES TB, T AKAYANAGI R, N AWATA H. (2007 B ). G A TRAZINE

- INDUCED AROMATASE EX PRESSION IS SF

- 1 DEPENDENT : IMPLICATIONS FOR

ENDOCRINE DISRUPTION IN WILDLIFE AND REPR ODUCTIVE CANCERS IN HUMAN S

. E NVIRON H EALTH P ERSPECT

. 115(5):720

- 7.

........ 88 A.4.5 H ENEWEER M, VAN DEN B ERG M, S ANDERSON JT. (2004). A COMPARISON OF HUMAN H295R A ND RAT R2C CELL LINES

AS IN VITRO SCREENIN G TOLLS FOR EFFECTS ON AROMATASE

. T OXICOL

. L ETT

. 149(2):183

- 194.

................................ ............... 88 A.4.6 H IGLEY EB, N EWSTED JL, Z HANG X, G IESY JP, H ECKER M. (2010). A SSESSMENT OF CHEMICA LS EFFECTS ON AROMAT ASE

ACTIVITY USING THE H295R CELL LINE

. E NVIRON S CI P OLLUT R ES I NT

. J AN 20. (E PUB AHEAD OF PRINT ).

................................ .... 90 A.4.7 H OLLOWAY , A. A NGER , D., C RANKSHAW , D., W U , M, AND F OSTER , W. 2008. A TRAZINE

- INDUCED C HANGES IN A ROMATASE A CTIVITY IN E STROGEN S ENSITIVE T ARGET T ISSUES

. J OURNAL OF A PPLIED T OXICOLOGY

. 28:2 60

- 270.

...................... 93 A.4.8 L AVILLE N, B ALAGUER P, B RION F, H INFRAY N, C ASELLAS C, P ORCHER JM, A IT

- A ISSA S. (2006). M ODULATION OF

AROMATASE AC TIVITY AND M RNA BY VARIOUS SELECTED PESTICIDES IN THE HU MAN CHORIOCARCINOMA JEG

- 3 CELL LINE

. T OXICOLOGY

. 228:98

- 108.

................................ ................................ ................................ ................................ .......... 93 A.4.9 R AYN ER , J., W OOD , C., AND F ENTON , S. (2004) E XPOSURE P ARAMETERS N ECESSARY FOR D ELAYED P UBERTY AND M AMMARY G LAND D EVELOPMENT IN L ONG

- E VANS R ATS E XPOSED IN U TERO TO A TRAZINE

. T OXICOLOGY AND A PPLIED P HARMACOLOGY 195:23

- 34.

................................ ................................ ................................ ................................ ....... 94 A.4.10 S UZAWA , M AND I NGRAHAM , HA. 2008. T HE HERBICIDE ATRAZIN E ACTIVATES ENDOCRIN E GENE NETWORKS VIA NON

STEROIDAL NR5A NUCLEAR RECEPTORS IN FISH AND MAMMALIAN CELLS

. PL O S ONE 3(5): E 2117.

DOI :10.1371/ JOURNAL

. PONE .0002117.

................................ ................................ ................................ ....................... 96 A.4.11 Y I D K, K IM P J, ET AL

. 2010. D OSE

- AND TIME

- DEPENDE NT EFFECT OF ATRAZIN E OR ITS METABOLITES ON AROMATASE

M RNA IN VITRO

. O NLY A PREPRINT OF AR TICLE AVAILABLE

. ................................ ................................ ................................ 97 A.4.12 Y I , KD, P ER EZ , EJ, B RECKENRIDGE , CB, AND S IMPKINS , JW (2010?) E X VIVO EFFECTS OF AT RAZINES OR METABOLIT ES

ON PHOSPHODIESTERASE ACTIVITY

. ................................ ................................ ................................ ................................ .. 98 A. 4.13 P OGRMIC

- M AJKIC K, F A S., D AKIC V, K AISAREVIC S., K OVACEVIC R. 2010. U PREGULATION OF P ERIPUBERTAL L EYDIG C ELL S TEROIDOGENESIS F OLLOWING 24 H I N V ITRO AND I N V ICO E XPOSURE TO A TRAZINE

. T OXICOL

. S CI

. 118(1): 52

- 60

..................... 99

A.5 NEUROTOXICITY

................................ ................................ ................................ ................................ ......... 101
Summary: Neu rotoxicity

................................ ................................ ................................ ................................ .. 102 A.5.1 B ELLONI V, A LLEVA E, ET AL

. 2007. E FFECTS OF LOW DOSES OF ATRAZINE ON THE N EUROBEHAVIOURAL DEVE LOPMENT OF

MI CE

. E THOLOGY E COLOGY & E VOLUTION 19: 309

– 322.

................................ ................................ ............................. 105 A.5.2 C OBAN A, AND F ILIPOV , N M. 2007. D OPAMINERGIC TOXICITY ASSOCIATED WITH ORAL EXPOSURE TO THE HERB ICIDE

ATRAZINE IN JUVENILE MALE C57BL/6 MICE

. J OURNAL O F N EUROCHEMISTRY 100: 1177

- 1187.

................................ ........... 107 A.5.3 F ILIPOV N M, S TEWART M A ET AL

. 2007. D OPAMINERGIC TOXICITY OF THE HERBICIDE ATR AZINE I N RAT STRIATAL SLICE S

. T OXICOLOGY 232: 68

– 78.

................................ ................................ ................................ ................................ ....... 108 A.5.4 F ORADORI , C D, H INDS , L R, ET AL

. 2009. A TRAZINE INHIBITS PUL SATILE LUT EINIZING HORMONE REL EASE WITHOUT

ALTERING PITUITARY S ENSITIVITY TO A GONA DOTROPIN

- RELEASING HORMONE RE CEPTOR AGONIST IN FE MALE WISTAR RATS

. B IOLOGY

OF R EPRODUCTION

. 81: 40

– 45.

................................ ................................ ................................ ............................... 109 A.5.5 F ORADORI , C D, H INDS , L R, ET AL

. 2009. E FFECTS OF ATRAZINE O N G N RH NEUROENDOCRINE FUNCT ION AFTER ITS

WITHDRAWAL IN THE AD ULT FEMALE WISTAR RA T

. B IOLOGY OF R EPRODUCTION 81(6): 1099

– 1105.

................................ ..... 110 A.5.6 G IUSI G, F ACCIOLO RM, ET AL

. 2006. T HE ENDOCRINE DISRUPT OR ATRAZINE ACCOUNTS FOR A DIMORPHIC

SOMATOSTATINERGIC NE URONAL EXPRESSION PATTERN I N MICE

. T OXICOLOGICAL S CIENCES 89(1): 257

– 264.

........................ 112 A.5.7 H OSSAIN M M AND F ILIPOV N M. 2008. A LTERATION O F DOPAMINE UPTAKE IN TO RAT STRIATAL VESI CLES AND

SYNAPTOSOMES CAUSED BY AN IN VITRO EXPOS URE TO ATRAZINE AND SOME OF ITS METABOLI TES

. T OXICOLOGY 248: 52

– 58.

.... 114Page 5 of 210 A.5.8 R ODRIGUEZ V M, T HIRUCHELVAM M, C ORY

- S LECHTA D A. 2005. S USTAINED E XPOSURE TO THE WIDEL Y USED HERBICIDE

ATRAZINE : ALTERED FUNCTION AND LOSS OF NEURONS IN B RAIN MONOAMINE SYSTE MS

. E NVIRONMENTAL H EALTH P ERSPECTIVES

. 113(6): 708

- 715.

................................ ................................ ................................ ................................ ................... 115 A 5.9 S OLARI , M, P AQUIN , J, D UCHARME , P, B OILY , M (2010). P19 NEURONAL DIFFERENTIA TION AND RETINOIC AC ID ME TABOLISM

AS CRITERIA TO INVES TIGATE ATRAZINE , NITRITE , AND NITRATE DEVELOPM ENTAL TOXICITY

. T OXICOL S CI 113 (1):116

- 126.

.......... 117 A.5.10 B ELLONI V, DESSI

- F ULGHERI , F, ET AL

. 2010. E ARLY EXPOSURE TO LOW DOSES OF ATRAZINE AF FECTS BEHAVIOR IN JU VENILE

AND ADULT CD1 MICE

. T OXICOLOGY (2010), DOI :10.1016/ J

. TOX .2010.07.002

................................ ............................... 117

A.6 IMMUNOTOXICITY

................................ ................................ ................................ ................................ ...... 121
Summary: Immunotoxicity

................................ ................................ ................................ .............................. 121 A.6.1 C IMINO

- R EALE , G., F ERRARIO , D., C ASATI , B., B RUSTIO , R. D IODOVICH , C., C OLLOTTA , A., V AHTER , M., G RIBALDO , L. (2008). C OMBINED IN UTERO AND JUVENILE EXPOSURE OF MICE AND ATRAZINE IN DRINKING WATER MODUL ATES GENE EXPRESSION

AND CLONOGENICITY OF MYELOID PROGENITORS

. T OXICOL

. L ETTERS 180:59

- 66.

................................ ................................ . 124 A.6.2 C ODER , P. S. (2010) A N O RAL (G AVAGE ) I MMUNOTOXICITY AND H ORMONE E VALUATION S TUDY FOR THE E FFECTS OF A TRAZINE ON M ALE R ATS , WIL R ESEARCH L ABORATORIES , LLC, 1407 G EORGE R OAD , A SHLAND , OH. U NPUBLISHED

. (TK0023175)

................................ ................................ ................................ ................................ ................................ .............. 125 A.6.3 D EVOS S, B OSSCHER K D E , ET AL

. 2003. I NHIBITION OF CYTOKIN E PRODUCTION BY THE HERBICIDE ATRAZINE

– S EARCH FOR

NUCLEAR RECEPTOR TAR GE TS

. B IOCHEMICAL P HARMACOLOGY 65: 303

– 308.

................................ ................................ ... 128 A.6.4 D EVOS S, V AN D EN H EUVEL R, ET AL

. 2004. L IMITED EFFECT OF SEL ECTED ORGANIC POLLUTANTS ON CYTOKI NE

PRODUCTION BY PERIPH ERAL BLOOD LEUKOCYT ES

. E UR

. C YTOKINE N ETW : 15: 145

– 151.

................................ .................. 130 A.6.5 F ILIPOV , N M, P INCHUK , L M, ET

. AL

. 2005. I MMUNOTOXIC EFFECTS O F SHORT

- TERM ATRAZINE EXPOSU RE IN YOUNG

MALE C57BL/6 MICE

. T OXICOLOGICAL S CIENCES : 86(2): 324

- 332.

................................ ................................ ................ 130 A.6.6 K ARROW , N A, M C C AY , J A, ET

. AL

. 2005. O RAL EXPOSURE TO ATRA ZINE MODULATES CELL

- MEDIATED IMMUNE FUNC TION

AND DECREASES HOST R ESISTANCE TO THE B16F10 TUMOR MODEL IN FEMAL E B6C3F1 MICE

. T OXICOLOGY : 15

- 28.

................. 131 A.6.7 M IZOTA K AND U EDA H. 2006. E NDOCRINE DISRUPTING CHEMICAL ATRAZINE CA USES DEGRANULATION T HROUGH G Q /11

PROTEIN

- COUPLED NEUROSTEROID RECEP TOR IN MAST CELLS

. T OXICOLOGICAL S CIENCES 90(2): 362

– 368.

............................ 132 A.6.8 P INCHUK L M, L EE S

- R, AND F ILIPOV N M. 2007. I N VITRO ATRA ZINE EXPOSURE AFFECT S THE PHENOTYPIC AND

FUNCTIONAL MATURATIO N OF DENDRITIC CELLS

. T OXICOLOGY AND A PPLIED P HARMACOLOGY 223: 206

– 217.

........................ 134 A.6.9 P RUETT , S B, F AN , R, ET AL

. 2003. M ODELING AND PREDICTI NG IMMUNOLOGICAL EFF ECTS OF CHEMICAL STR ESSORS :
CHARACTERIZATION OF A QUANTITATIVE BIOMA RKER FOR IMMUNOLOGIC AL CHANGES CAUSED BY ATRAZINE AND ETHANOL

. T OXICOLOG ICAL S CIENCES : 75: 343

- 354.

................................ ................................ ................................ .................... 134 A.6.10 P RUETT , S B, F AN , R, ET AL

. 2009. P ATTERNS OF IMMUNOTOX ICITY ASSOCIATED WIT H CHRONIC AS COMPARED WITH ACUTE

EXPOSURE TO CHEMICAL OR PHYSICAL STRESSOR S AND THEIR RELEVANC E WITH REGARD TO THE ROLE OF STRESS AND W ITH REGARD TO

IMMUNOTOXICITY TESTI NG

. T OXICOLOGICAL S CIENCES : 109(2): 265

- 275.

................................ ................................ ........ 136 A.6.11 R AJKOVIC , V, M ATAVULJ , M AND J OHANSSON , O. (2010). C OMBINED E XPOSURE OF P ERIPUBERTAL M ALE R ATS TO THE E NDOCRINE

- D ISRUPTING C OMPOUND A TRAZINE AND P OWER

- F REQUENCY E LECTRO MAGNETIC F IELDS C AUSES D EGRANULATION OF C UTANEOUS M AST C ELLS : A N EW T OXIC E NVIRONMENTAL H AZARD ? A RCH

. E NVIRON

. C ONTAM

. T OXICOL

. DOI 10:1007/ S 00244

- 010

- 9477

- 6

................................ ................................ ................................ ................................ ............................ 137 A.6.12 R OONEY , A A, M ATULKA R, AND L UEBKE , R. 2003. D EVELOPMENTAL ATRAZIN E EXPOSURE SUPPRESSE S IMMUNE FUNCTION

IN MALE , BUT NOT FEMALE S PRAGUE

- D AWLEY RATS

. T OXICOLOGICAL S CIENCES 76: 366

- 375.

................................ ............... 138 A.6.13 R OWE , A M, B RUNDAGE , K M, S CHAFER , R, AND B ARNETT J B. 2006. I MMUNOMODULATORY EFFE CTS OF MATERNAL

ATRAZINE EXPOSURE ON MALE B ALB / C MICE

. T OXICOLOGY AND APPLIE D P HARMACOLOGY 21: 69

– 77.

................................ .. 139 A.6.14 R OWE A M, B RUNDAGE K M AND B ARNETT J B. 2007. I N VITRO ATRAZINE

- EXPO SURE INHIBITS HUMAN NATURAL KILLER

CELL LYTIC GRANULE R ELEASE

. T OXICOLOGY AND A PPLIED P HARMACOLOGY 221: 179

– 188.

................................ ................ 141 A.6.15 S C HWAB , C L, F AN R, ET AL

. 2005. M ODELING AND PREDICTI NG STRESS

- INDUCED IMMUNOSUPPRE SSION IN MICE USING

BLOOD PARAMETERS

. T OXICOLOGICAL S CIENCES 83: 101

– 113.

................................ ................................ ...................... 141 A.6.16 W HALEN M M, L OGANATHAN B G, ET AL

. 2003. I MMUNOMODULATION OF H UMAN NATURAL KILLER CELL CYTOTOXIC

FUNCTION BY TRIAZINE AND CARBAMATE PESTIC IDES

. C HEMICO

- B IOLOGICAL I NTERACTIONS 145: 311

– 319.

.......................... 142
A.7 CARCINOGENESIS/ GENOTOXICITY

................................ ................................ ................................ .............. 142 Summary: Carcinogenesis/Genotoxicity

................................ ................................ ................................ ......... 142 A.7.1 F UJIMOTO , N AND H ONDA , H. (2003). E FFECTS OF E NVIRONMENTAL E STROGENIC C OMPOUNDS ON G ROWTH OF A T RANSPLANTED E STROGEN R ESPONSIVE P ITUITARY T UMOR C ELL L INE IN R ATS

. F OOD C HEM

. T OXICOL

. 41:1711

- 1717.

............ 145Page 6 of 210 A.7.2 F UKAMACHI , K., H AN , B.S., K IM , C.K., T AKASUKA , N., M ATSUOKA , Y., M ATSUDA , E., Y AMASAKI , T., AND T SUDA , H. (2004). P OSSIBLE ENHANCING EF FECTS OF ATRAZINE AN D NONYLPHENOL ON 7,2

- DIMETHYLBENZ [ A ] ANTHRACENE

- INDUCED MAMMARY

TUMOR DEVELOPMENT IN HUMAN C

- H A

- RAS PROTO

- ONCOGENE TRANSGENIC RATS

. C ANCER S CI

. 95(5):404

- 410.

.................... 146 A.7.3 G ARAJ

- V RHOVAC , V. AND Z EIJEZIC , D. (2004) G ENOTOXICITY EVALUATI ON OF PESTICIDE FORM ULATIONS CONTAINING

ALACHLOR AND ATRAZIN E IN MULTIPLE MOUSE TISSUES ( BLOOD , KIDNEY , LIVER , BONE MARROW , SPLEEN ) BY COMET ASSAY

. N EOPLASMA 5 1.3:198

- 203.

................................ ................................ ................................ ................................ ...... 147 A.7.4 K METIC , I., S RCEK , G., S LIVAC , I., S IMIC , B., K NLEWALD , Z., K NIEWALD , J. 2008. A TRAZINE E XPOSURE D ECREASES C ELL P ROLIFERATION IN C HINESE H AMSTER O VARY (CHO

- K1) C ELL L INE

. B ULL E NVIRON C ONTAM T OXICOL

. 81: 205

- 209.

.............. 149 A.7.5 L ASSERRE , JP
, F ACK , F, R EVETS , D, P LANCHON S, R ENAUT J, H OFFMANN L, G UTLEB , M ULLER CP, B OHN T, (2009) E FFECTS

OF THE E NDOCRINE D ISRUPTORS A TRAZINE AND PCB 153 ON THE P ROTEIN E XPRESSION OF MCF

- 7 H UMAN C ELLS

. J OURNAL OF P ROTEOME R ESEARCH

. ................................ ................................ ................................ ................................ ............... 151 A.7.6 M ALIK , S. I., T ERZOUDI , G.I., AND P ANTELIAS , G.E. (2004). SCE ANALYSIS IN G2 LYMPHOCYTES PREMATUR ELY

CONDENSED CHROMOSOME S AFTER EXPOSURE TO ATRAZINE : THE NON

- DOSE

- DEPENDENT INCREASE I N HOMOLOGOUS

RECOMBINATIONAL EVEN TS DOES NOT SUPPORT ITS GENOTOXIC MODE O F ACTION

. C YTOGENET G ENOME R ES 104: 315

- 319.

..... 152 A.7.7 M ANSKE , M., B ELTZ , L, AND D HANWADA , K. 2004. L OW

- L EVEL A TRAZINE E XPOSURE D ECREASES C ELL P ROLIFERATION IN H UMAN F IBROBLASTS

. A RCH

. E NVIRON

. C ONTAM

. T OXICOL

. 46:438

- 444.

................................ ................................ ......... 153 A.7.8 M ASTRANGELO , S., T OMASSETTI , M, C ARRATU , M.R., E VANDRI , M.G., AND B OLLE , P. (2006). Q UERCETIN REDUCES

CHROMOSOME ABERRATIO NS INDUCED BY ATRAZI NE IN THE A LLIUM CEPA TEST

. E NVIRO N AND M OL M UTAGEN 47:254

- 259.

...... 155 A.7.9 O LEJNIK , A M , M ARECIK , R, B IALAS , W, C YPLIK , P. (2010). I N VITRO STUDIES ON A TRAZINE EFFECTS ON HUMAN INTESTINAL

CELLS

. W ATER A IR S OIL P OLLUT

. DOI 10.1007/ S 11270

- 010

- 0394

- 0; PUBLISHED INLINE : A PRIL 16, 2010

......................... 156 A.7.10 S INGH , M, S ANDHIR , R, K IRAN , R AVI

. (2010). E FFECTS ON ANTIOXIDAN T STATUS OF LIVER FO LLOWING ATRAZINE EXP OSURE

AND ITS ATTENUATION BY VITAMIN E. E XP AND T OXICOL P ATHOL DOI :10.1016/ JETP .2010.01.005

................................ ....... 1 58 A.7.11 S INGH , M., S ANDHIR , R., AND K IRAN , R. 2008. A TRAZINE

- I NDUCED A LTERATIONS IN R AT E RYTHROCYTE M EMBRANES : A MELIORATING E FFECT OF V ITAMIN E. J. B IOCHEM

. M OLECULAR T OXICOLOGY

. V OL 22, N O

. 5: PGS

. 363

- 369.

........................ 159 A.7.12 S INGH , M., K AUR , P., S ANDHIR , R., K IRAN , R. (2008). P ROTECTIVE EFFECT OF VITAMIN E AGAINST ATRAZI NE

- INDUCED

GENOTOXICITY IN RATS

. M UTAT R ES 654:145

- 149.

................................ ................................ ................................ ....... 160 A.7.13 S ON , H

- Y., N ISHIKAWA , A., O KAZAKI , K., L EE , K

- Y., I MAZAWA , T., AND H IROSE , M. (2003). L ACK OF M ODIFYING E FFECTS

OF A TRAZINE AND / OR T AMOXIFEN ON T HYROID C ARCINOGENESIS IN R ATS P RETREATED WITH N

- BIS (2

- HYDROXYPROPYL ) NITROSAMINE (DHNP). F OOD C HEM

. T OXICOL

. 41:1811

- 1816.

................................ ................................ ................................ ........ 161 A.7.14 T ANAKA , T., K OHNO , H., S UZUKI , R., S UGIE , S. (2004) L ACK OF MODIFYING EFF ECTS OF AN ESTROGENI C COMPOUND

ATRAZINE ON 7,12

- DIMETHYLBENZ ( A ) ANTHRACENE

- INDUCED OVARIAN CARC INOGENESIS IN RATS

. C ANCER L ETT

. 210:129

- 137.

. 162 A.7.15 U EDA , M., I MAI , T., T AKIZAWA , T., O NODERA , H., M ITSUMORI , K., M TSUI , T. AND H IROSE , M. (2005) P OSSIBLE

ENHANCING EFFECTS OF ATRAZINE ON GROWTH O F 7,12

- DIMETHYLBENZ ( A ) ANTHRACENE

- INDUCED MAMMARY TUMO RS IN

OVARIECTOMIZED S PRAGUE

- D AWLEY RATS

. C ANCER S CI

. 96(1):19

- 25.

................................ ................................ .............. 163 A.7.16 Y I , K UN D ON , B RECKENRIDGE , C HARLES B, AND S IMPKINS , J AMES W. 2010. C YTOTOXICITY OF A TRAZINE AND ITS M ETABOLITES IN VITRO

. U NPUBLISHED DATA

. S YNGENTA C ROP P ROTECTION I NC

. ................................ ................................ .. 167
A.8 INSULIN/MITOCHO NDRIA FUNCTION

................................ ................................ ................................ ......... 168 Summary: Insulin/Mitochondria Function

................................ ................................ ................................ ...... 168 A.8.1 L IM , S., A HN , S.Y., S ONG , I.C., C HUNG , M.H., J ANG , H.C., P ARK , K.S., L EE , K

- U, P AK , Y.K., L EE , H.K. (2009). C HRONIC E XPOSURE TO THE H ERBICIDE , A TRAZINE , C AUSES M ITOCHONDRIAL D YSFUNCTION AND I NSULIN R ESISTANCE

. PL O S ONE 4(4):

E 5186, P .1

- 11.

................................ ................................ ................................ ................................ ........................ 169

A.9.1. PHARMACOKINET IC CALCULATIONS

................................ ................................ ................................ ........ 170

A.9.2. PHARMACOKINETIC STUDIES

- MINIREVIEWS

................................ ................................ ............................. 199 A Physiologically Based Pharmacokinetic (PBPK) Model for Atrazine and its Main Metabolites in the Adult Male C57BL/6 Mouse Toxicology and Applied Pharmacology 251 (2011) 16

- 31

................................ ............. 199 In Vitro Metabolism of Atrazine in Rat and Human Hepatocytes

................................ ................................ .... 201 PBPK Model for Atrazine and its Chlorinated Metabolites

................................ ................................ ............... 201

A. 10 MIXTURES

................................ ................................ ................................ ................................ ................. 202Page 7 of 210 A.10.1 E NOCH , R.R., S TANKO , J.P., G REINER , S.N., Y OUNGBLOOD , G.L. R AYNER , J.L., F ENTON , S.E., 2007. M AMMARY G LAND D EVELOPMENT AS A S ENSITIVE E ND P OINT AFTER A CUTE P RENATAL E XPOSURE TO AN A TRAZINE M ETABOLITE M IXTURE IN F EMALE L ONG

- E VANS R ATS

. E NVIRONMENTAL H EALTH P ERSPECTIVES

. 115(4): 541

- 547.

................................ .............................. 202

A.11 OTHER

................................ ................................ ................................ ................................ ....................... 207

A.11.1 F AKHOURI , WD, N UNEZ , JL, T RAIL , F. (2010?) A TRAZINE B INDS TO THE G ROWTH H ORMONE R ELEASING H ORMONE R ECEPTOR AND A FFECTS G ROWTH H ORMONE G ENE E XPRESSION

. I N PRESS

................................ ................................ .......... 207 A.11.2 Y I , KD, P EREZ , EJ, B RECKENRIDGE , CB, AND S IMPKINS , JW (2010) E X VIVO EFFECTS OF AT RAZINES OR METABOLIT ES ON

PHOSPHODIESTERASE AC TIVITY

. I N PRESS

................................ ................................ ................................ ........................ 209Page 8 of 210 A.1 CONTENT OF THIS APPENDIX

An evaluation of peer

- reviewed articles related to atrazine and/or its metabolites published since 2003 was conducted in preparation for the April 2010 SAP and updated prior to the September meeting. The following table provides a listing of the new stud ies available between January 30 and July 15, 2010 (deadlines for the April and September meetings, respectively). The scope of this evaluation was restricted to articles reporting on mammalian toxicity studies or in vitro studies that provided data on at razine and atrazine metabolites mode of action (MOA). The purpose of this evaluation was to determine the scientific quality of the research conducted since the atrazine Re

- registration Eligibility Decision (RED) was finalized in 2003. Ultimately, the ob jective of this evaluation is to ascertain if these new data impact the Agency’s conclusions regarding points of departure, MOA and overall impact of atrazine exposure on human health. To the extent that these data provide additional information on these areas they will be used to update the atrazine human health risk assessment.

Author/Title Section Coder P. (2011e). Atrazine: An Oral (Gavage) Study of the Effects of Atrazine on the Estrous Cycle and the Estrogen

- Induced Luteinizing Hormone Surge in Female Rats. Final Report. Replaces MRID 48180103) Unpublished data from Syngenta Crop Protection, Inc. Study completion date: February 4, 2011. Report Number: WIL

- 639060. MRID 48381001 A.2.4

Davis LK, Murr AS, Best DS, Fraites MJP, Zorrilla LM, Marotsk y MG, Stoker TE, Goldman JM, Cooper RL. 2011. The Effects of Prenatal Exposure to Atrazine on Pubertal and Postnatal Reproductive Indices in the Female Rat. Reprod. Toxicol., doi:10.1016/j.reprotox.2011.04.004 A.2.15

Goldman J, Davis L, Murr A, Cooper R. (2011). The Influence of 1, 2, and 4 Days of Atrazine Treatments on the LH Surge in Ovariectomized/Estradiol

- Primed Rats: Preliminary Report to the Office of Chemical Safety and Pollution Prevention. Internal Report USEPA A.2.16

Coder, P. S. (2011) An Oral (Gavage and Dietary) Study of the Effects of Atrazine on the Spontaneous Luteinizing Hormone Surge in Female Sprague

- Dawley Rats , WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. Study Number: WIL

- 639080 A.2.17

Coder, P. S. (2011) An Study of the Effects of Short

- Term Atrazine Exposure on the Estrogen

- Induced Luteinizing Hormone (LH) Surge in Ovariectomized Young

- Adult Sprague Dawley Female Rats, WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. Stu dy Number: WIL

- 639091 A.2.18

Coder, P. (2011d) Atrazine: An Oral (Gavage and Dietary) Study of the Effects of Atrazine on Fertility and Reproductive Performance in Female Long Evans and Sprague

- Dawley Rats: Final Report. Report Number: WIL/639090, Task No. TK0052068. Unpublished study. Performing Laboratory: WIL Research Laboratories, Inc. 966 p. Completion Date: April 6, 2011. MRID 48470001. A.2.19

Fraites MJP, Narotsky MG, Best DS, Stoker TE, Davis LK, Goldman JM, Hotchkiss MG, Klinefelter GR, Kamel A , Qian Y, Podhorniak L, Cooper RL. (2011). Gestational Atrazine Exposure Effects on Male Reproductive Development and Metabolite Distribution in the Dam, Fetus, and Neonate. Reprod. Toxicol., doi:10.1016/j.reprotox.2011.04.003 A.3.17

Pogrmic

- Majkic K, Fa S., Dakic V, Kaisarevic S., Kovacevic R. 2010. Upregulation of Peripubertal Leydig Cell Steroidogenesis Following 24 h **In Vitro** and **In Vico** Exposure A.4.13Page 9 of 210 to Atrazine. **Toxicol. Sci.** 118(1): 52

- 60 Belloni V, dessi

- Fulgheri, F, et al. 2010. Early exposure to low doses of atrazine affects behavior in juvenile and adult CD1 mice. Toxicology (2010), doi:10.1016/j.tox.2010.07.002 A.5.10

Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L. Rayner, J.L., Fenton, S.E., 2007. Mammar y Gland Development as a Sensitive End Point after Acute Prenatal Exposure to an Atrazine Metabolite Mixture in Female Long

- Evans Rats. Environmental Health Perspectives. 115(4): 541

- 547.

Stanko, J., Enoch, R., Rayner, J., Davis, C., Wolf, D., Malarkey, D., and Fenton S. 2010. Effects of Prenatal Exposure to a Low Dose Atrazine Metabolite Mixture on Pubertal Timing and Prostate Development of Male Long Evans Rats. Reproductive Toxicology. 30:540

- 549. A.10.1Page 10 of 210 A.2 EFFECTS ON FEMA LES

Summary: Vaginal Opening, Ovarian Function

.

Over the last seven years, studies have been conducted to evaluate the impact of atrazine and/or its by

- products on the development and reproductive health of females. 1 In 2003 Laws et al., evaluated the effects of DACT and hydroxyatrazine (a plant and bacterial metabolite of atrazine) on the pubertal development of female Wistar rats. This study is a follow

- up to the researchers’ previous findings that 20 days of exposure (PND 22

- 41) to the parent compound

– atr azine

– at doses ≥ 50 mg/kg/day induced a delay in female sexual maturation manifested as a delay in vaginal opening (VO) (Laws et al., 2000). DACT exposure at high doses ( ≥ 34 mg/kg/day) from post

- natal day PND 22

- 41 resulted in significant delays in VO (3

- 7 day delay) and in some instances animals failed to attain VO by the time of their scheduled sacrifice on PND 41. Interestingly, these delays in VO were not accompanied by lower body weights thus they do not appear to be related to generalized growth delays. Moreover, the NOAEL identified for DACT is the same as equimolar equivalent NOAEL for atrazine. In contrast, hydroxyatrazine exposure at doses up to 183 mg/kg/day did not result in delays in VO. Given that atrazine is quickly metabolized to DACT , these results strongly suggest that DACT is the toxicologically active moiety causing the delays in sexual maturation induced by atrazine.

Potential effects of atrazine on ovarian function were evaluated over a wide dose range (3

- 300 mg/kg/day) by Shi bayama and colleagues (Shibayama et al., 2009). In a companion study these researchers attempted to correlate atrazine

- induced changes in ovarian function to fertility impairments. Unfortunately, different dose levels were used for the ovarian function a nd fertility studies thus precluding an appropriate comparison between these two sets of experiments. In the ovarian function set of experiments, atrazine exposure for 2 or 4 weeks at a high dose (300 mg/kg/day) resulted in irregular estrous cycles (typica lly longer cycles) due primarily to a lengthened diestrus. This effect was accompanied by decreased numbers of corpora lutea, follicular atresia, uterine atrophy, as well as decreased ovarian and uterine weights. Noticeably, the duration of atrazine expo sure (2 vs. 4 weeks) had no effect on the nature, severity, or dose level causing the estrous cycle disruption or the histopathology changes. In the fertility study, atrazine exposures at levels up to 100 mg/kg/day for a period of time encompassing 2 week s prior to mating up to gestation day (GD) 7 did not result in any signs of impaired fertility. None of the signs typically associated with impaired fertility ( e.g., number of implantation, corpora lutea, pre

- or post

- implantation loss) were affected.

Th e studies by Laws et al., and Shibayama et al., indicate that exposure to atrazine or DACT results in effects related to the development and function of the reproductive

1 Studies on the potential effect of atrazine exposure on mammary gland development will be evaluated during the Fall meeting of the SAP.Page 11 of 210 system. The effects described by both groups are consistent with the postulated MOA

o f perturbations of the neuroendocrine system. Sexual maturation is the culmination of a complex cascade of sex developmental effects that ultimately leads to the attainment of reproductive capacity

. Activation of the HPG axis resulting in the pulsatile s ecretion of GnRH and LH is critical to puberty onset. Researchers have found that disruption of GnRH release can lead to delays in vaginal opening. Thus, it is not unexpected that exposure to atrazine and DACT

– atrazine’s metabolite

– leads to a delay i n sexual maturation given atrazine’s effect on the HPG axis. Similarly, the lengthening of the diestrus stage after atrazine exposure can be readily explained by the disruptions to the HPG axis caused by atrazine. Even through the relatively short period of time evaluated by Shibayama et al., it is clear that atrazine treatment leads to a state of reproductive quiescence which

– if sustained

– would eventually result in the reproductive senescence characteristic of repeated atrazine exposure. It is notew orthy that the NOAEL for the effects reported after ≈ 3

- 4 weeks of exposure by Laws et al. (2000)

, and Shibayama et al., (25 and 30 mg/kg/day, respectively in atrazine molar equivalents) were higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge identified after a 6

- month exposure and that is currently used as the point of departure in the Agency’s risk assessment.

Summary: Mammary Gland Development

. Mammary gland development is a complex biological process controlled by multiple hormonal interactions. The interplay of pituitary, ovarian, and adrenal hormones regulate the development, differentiation, and proliferation in mammary glands (Daniel et al ., 1987; Imagawa et al., 1990) . Over the last century, extensive research has been condu cted to understand the underlying biological processes involved in mammary gland development and the hormonal signals and pathways that modulate it (Hennighausen and Robinson, 2001). In a series of seminal experiments in 1900, Haban first described the ro le ovaries played in mammary gland development by demonstrating that in ovariectomized animals mammary gland tissue regressed and that transplanting ovaries back into these animals prevented this atrophy. In the 1920s several investigators showed that the pituitary gland was also involved in mammary gland development. Thus, began the era of mammary gland development research.

Development of mammary tissue occurs in defined stages linked to sexual maturation and reproduction including embryonic, pre

- an d peripubertal periods, as well as pregnancy and lactation. Given the effects atrazine has on the hormonal environment and reproductive systems, it is not unreasonable to hypothesize that it may also affect mammary gland development. A number of studies have been conducted to investigate this possibility. Several studies from the same research group (Rayner et al., 2004; Rayner et al. , 2005; and Enoch et al. , 2007 [see mixtures section below]) describe effects of either atrazine or a mixture of atrazine metabolites/degradates (DIA, DEA, DACT, hydroxy

- atrazine) on mammary gland development in female pups. Briefly, the authors concluded that atrazine delays mammary gland development using a subjective scoring scale to evaluate mammary gland development.The Agency’s reviews of these studies are summarized in this appendix. The Agency concluded that there arePage 12 of 210 shortcomings in the study design, data reporting and analysis that compromise the interpretation of the studies.

A fourth study recently submitted t o the Agency also assessed the impact of atrazine exposure on mammary gland development (Coder, 2010). Animals were exposed in utero during the presumptive critical period for mammary gland development (GD 13

- 19) to atrazine at doses ranging from 6.5

- 100 mg/kg/day. In contrast to the Rayner et al

. and Enoch et al. studies that used a subjective scoring scale to assess mammary gland development, Coder employed an objective measure (morphometry) to evaluate mammary gland development. Unlike the previous st udies, the Coder data suggest that atrazine does not affect mammary gland development. The Agency’s review of this study is also included in this appendix.

A.2.1 Casas, E., Bonilla, E., Ducolomb Y, Betancourt M. 2010. Differential effects of herbicides a trazine and fenoxaprop

- ethyl, and insecticides diazinon and malathion, on viability and maturation of porcine oocytes **in vitro** . Toxicology **in vitro**  24 (2010); 224

- 230.

The purpose of this study was to evaluate the effect of two commercially

- formulated ins ecticides (malathion and diazinon) and two technical grade herbicides (atrazine and fenoxaprop

- ethyl) on viability and in vitro maturation of porcine oocytes. **Only the results of atrazine will be discussed in this summary.** 

Ovaries were collected from slaughtered 6

- month old gilts and oocytes were matured in increasing concentrations of atrazine for 44 hours. Atrazine was added in 0, 50, 100 and 500 μM concentrations. Viability and in vitro maturation were evaluated simulta neously in the same oocyte. After in vitro maturation, the oocytes were stained with methylthiazolydiphenyl

- tetrazolium bromide (MTT) for 2 hours to assess viability. To assess maturation, oocytes were treated with 10 ug/ml bisbenzimide. Purple stained oocytes were considered alive when evaluated under a light microscope. Oocytes having a germinal vesicle (GV) were considered immature. Oocytes in first metaphase (MI) were judged to be maturing. Those showing a second metaphase (MII) and a polar body were classified as matured. The correlation among pesticide concentration and its effect on ooctye viability and maturation was assessed using Spearman’s rank correlation coefficient. The concentration required to produce death in 50% of the oocytes (lethal concentration 50, LC 50 ) and the concentration needed to inhibit the maturation in 50% of the oocytes (inhibition of maturation 50, IM 50 ) were calculated by a non

- linear regression.

For atrazine, 664 oocytes (total) were analyzed in five assays at 0, 50, 100 and 500 μM atrazine concentrations. For viability, the Spearman correlation was not significant, therefore, it was not possible to calculate the LC 50 . For maturation, the Spearman correlation was significant and the estimated IM 50 was 143 μM. Exposu re to 100 and 500 μM atrazine significantly increased (p<0.05) the percentage of oocytes in the GV stage to 40% and 79%, respectively, compared to controls (16%). The MI values werePage 13 of 210 not affected at any dose level. The percentage of mature oocytes (MII) decreased at 100 μM (33%) and 500 μM (2%), compared to controls (61%).

Reviewer’s Comments: Atrazine had an effect on maturation but not on viability, due to a blockage at the germinal vesicle stage.

A.2.2 Coder, P.S. 2010. An Oral Developmental Toxic ity Study of Atrazine in the Rat, Including Cross

- Fostering and Pair

- Feeding. Unpublished Syngenta study

The purpose of this study was to assess the impact of atrazine on: (i) mammary gland development; (ii) in utero only or lactational only exposure; as well as the effect of maternal food consumption on the offspring.

Long

- Evans dams received atrazine (97.1% a.i.) from GD 13

- 19 [the presumptive critical period for mammary gland development] at doses of 0, 6.5, 50, and 100 mg/kg/day. The control and high

- dose groups contained 42 dams each while the low

and mid

- dose contained 18 dams). The larger number in the control and high dose group was to accommodate the need for rats used in the cross

- fostering portion of the study. Initially, 15 dams (and their litters) from the control and high dose groups were intended to be assigned to the cross

- fostering module of this study. However, due to maternal aggression and cannibalization of pups this portion of the study was terminated after only 8 litters were ass igned before this aspect of the study was terminated on PND 2. 2 An additional group of 18 dams was used for the pair

- feeding component of the study and their feed was restricted to match the food consumption of the 100 mg/kg/day rats. Litters were standar dized on PND 4 to 10 pups/litter (with as many females retained as possible). 3 In addition to the common assessments ( clinical observations, body weight and food consumption measures), all female pups were weighed on the day of vaginal opening (VO), and vaginal lavages performed daily to check for estrous cyclicity beginning on PND 50

- 72. One F 1 female pup/litter was sacrificed on PND 1, 21, 333, day of VO, first day of diestrous after PND 58 to conduct mammary gland evaluations. Mammary gland evaluatio ns consisted of morphometric analysis of whole mounted 4 th mammary gland. Two hours prior to sacrifice the dams were administered BrDU to assess for cell proliferation in the mammary tissue. Another F 1 female pup/litter was mated with untreated males to p roduce an F 2 generation and allowed to deliver and rear their offspring until PND 21. The F 2 pups were sexed and subjected to clinical observations, body weight and food consumption determinations.

For P dams

2 This aspect of the study will not be discussed further in this review. a slight but statistically significant decrea se in body weight was noted at the 50 and 100 mg/kg/day dose levels ( ↓4

- 13%) during gestation. Body weight gains were also statistically significantly (p<0.01) reduced during this period at 50 and 100 mg/kg/day ( ↓30 and 59%, respectively). Although overal l body weight gain during lactation was increased at the high dose ( ↑ 26%, p<0.05), the body weight at the mid

and high

- dose remained lower relative to control ( ↓4

- 11%) probably due to the initial

3 Only litters comprised of ≥ 6 female pups and at least 10 pups total were used for litter standardization and subsequent assessmentsPage 14 of 210 lower body weight because of the gestational body weight d ecrements. In general the body weight of the pair

- fed group animals was statistically significantly lower than control ( ↓33%) and the highest dose tested (↓47%). During gestation, decreases in food consumption and food efficiency were noted at doses ≥ 50 mg/kg/day ( ↓22

- 37% and 11

- 39%, respectively). Food consumption and food efficiency were either unaffected or increased during lactation. No significant clinical signs or necropsy findings were noted at any dose level. Also unaffected was the gestation length.

Total litter loss was increased at the 100 mg/kg/day dose level (21% vs. 9% control). For most dams this was attributed to diminished nursing behavior (no milk band on pups), poor nesting behavior and/or aggression towards pups. In addition, exp osure to 100 mg/kg/day atrazine resulted in decreases in litter size ( ↓11%), live litter size (↓15%), pup survival between PND 0

- 1 (76% survival vs. 91% control), litter weight ( ↓5

- 14%), and pup weight gain from PND 10

- 14 ( ↓16

- 19%, p<0.05). Sixty eight pu ps were sacrificed in extremis at the high dose (21 of these pups had no milk in their stomachs)

vs. 36 in control. No other findings were reported in the surviving F 1 pups. Once they reached adulthood, no effects were reported in F 1 animals. Similarly, F 2 pups were unaffected. 4

Mammary gland assessments were conducted on the 4 th mammary gland collected on PND 1, 21, 33, day of VO, and first diestrous after PND 58. The left mammary gland was used for whole mount and subjected to morphometric measuremen ts of the ductal length, ductal network area, epithelial area, and number of end buds. Three hundred seventy six specimens were collected but 38 could not be used (the unusable specimens were equally distributed among dose groups).The 4 th right mammary gl and was used for cell proliferation analysis using a BrDU immunoassay.

No end buds were present on PND1 animals and a few of the PND21 animals. On PND1 , a statistically significant (p<0.01) 20% increase on mammary ductal length was reported at doses ≥ 50 mg/kg/day along with a decrease in the proliferation of epithelial cells at 100 mg/kg/day ( ↓31%, p< 0.01). Though not statistically significant, an increase in the number of end buds ( ↑27%) was noted at the 100 mg/kg/day dose level on PND21. No effects were noted on PND 33 . However, on the day of VO an increase in the number of end buds was noted for the 50 and 100 mg/kg/day dose group as well as the offspring of the pair

- fed ( ↑32, 17, and 113%, respectively). No effects on mammary gland d evelopment were observed in animals sacrificed after PND 58.

Except for a decrease in pup survival from PND 4

- 21 (83% survival vs 98% in control), and an increase in the number of end buds on the day of VO, the offspring of pair

- fed animals exhibited litt le effects. The pair

- fed dams themselves had a decrease in body weight relative to control ( ↓7

- 16%, p<0.01) during GD 13

- 19. Body weight gain during the treatment period was also decreased relative to both control and the high

- dose group ( ↓78 and 45%, p< 0.01 respectively) as well as food efficiency ( ↓63%, p<0.01). During lactation, the body weight of pair fed dams was 16

- 25% lower than control and

4 F 1 and F 2 pups were never directly exposed to atrazine. Thus any effects seen in either of these generations would have been the result of in utero exposure of the F 1 generationPage 15 of 210 5

- 19% lower than the high

- dose. The body weight gain during this period was 33% lower than control and 45% lower than the high

- dose group while the food efficiency was 28% lower than control.

Reviewer Comments: In general, this is a well

- conducted study. One of the initial goals of this study was to evaluate the effect of atrazine exposure in utero only or d uring lactation only in the development of the offspring. This portion of the study had to be terminated on PND 2 due to maternal aggression and cannibalism. The other major purpose of the study was to conduct an objective and quantitative evaluation of mammary gland development after atrazine exposure during GD 13

- 19. With the exception of an increase in the mammary ductal length reported on PND 21 offspring, there were no effects on mammary gland development that could be attributed to in utero atrazin e exposure. Although there was an increase in the number of end buds observed on the day of VO in the 50 and 100 mg/kg/day dose levels relative to control, the increase in the number of end buds in the pair

- fed group was even larger suggesting that this e ffect may be more related to the decrease in body weight/body weight gain than to atrazine exposure per se . The NOAEL for both dams and their offspring is 6.5 mg/kg/day and the LOAEL is 50 mg/kg/day based on decreases in body weight, body weight gain, foo d consumption, and food efficiency (maternal animals) and an increase in mammary ductal length and decrease in epithelial cell proliferation on PND1 (offspring). It should be noted, however, that these putative effects on mammary gland development perinat ally did not result in any sustained effects that could be observed at any other time point in development. Thus their toxicological significance is equivocal. A.2.3 Coder, P.S. 2010. An Oral (Gavage) Study to Assess the Potential for Detecting the Est rogen - Induced Luteinizing Hormone Surge in Female Rats. Syngenta Report.

In this study the author attempted to replicate results previously reported by Cooper et al. regarding the impact of a brief (4

- day) exposure to atrazine on the estrogen

- induced LH surge in ovariectomized (OVXD) females.

Initially, 20 adult nulliparous Sprague Dawley rats were assigned to receive 0 or 100 mg/kg/day of atrazine (purity 98.8% a.i.) for 4 consecutive days after ovariectomy and implantation of an estradiol benzoate co ntaining capsule (4 mg/ml) 5

5 The estradiol benzoate implants produced a daily afternoon LH surge that yielded a synchronized cohort of animals that yielded a surge where the timing and amplitude of each surge could be readil y quantified . On study day 0 (first day of study), all females were OVXD and implanted with both an estradiol benzoate capsule and a femoral vein catheter (to allow rapid collection of multiple blood samples). Due to mortalities during or shortly after the surgical procedure (typically before the first atrazine treatment), animals were reassigned to ensure that at least 16 rats/group were available for hormone assessments. Thirty minutes after surgery animals were gavaged with 0 or 100 mg/k g/day of atrazine. Treatment was done each subsequent day at approximately the same time of day (1200 to 1549 hours). On the fourth day of treatment (study day 3), blood samples were collected at 1300, 1600, 1800, 2000, 2300, and 0100 (study day 4) hours to analyze for luteinizing hormone (LH)Page 16 of 210 and corticosterone blood levels. After the last sampling, animals were sacrificed as quickly as possible and a terminal blood sample was collected for the possible evaluation of plasma levels of estradiol, estriol , and estrone.

Five and 2 rats died prior to the first administration of atrazine in the 0 and 100 mg/kg/day group, respectively. One female in the 100 mg/kg/day group died after the first atrazine administration but necropsy indicated that this death was also due to po st

operative complications.

The incidence of wet yellow material in the urogenital area and right hindlimb was increased in the treatment group (13/16 and 10/16, respectively vs 1

- 2/16 control). Also increased in the 100 mg/kg/day group was the inciden ce of dried red material around the mouth (10/16 vs 2/16 control). A statistically significant (p< 0.01) decrease ( ↓6%) in body weight was reported at the end of the study for the treatment group. This is consistent with a marked decrease in food consump tion ( ↓54

- 250%, p<0.01).

LH and corticosterone plasma levels were measured using radioimmunoassay techniques. Peak amplitudes of LH were measured by determining the peak hormone concentration regardless of timing. The area under the curve (AUC) for the hormone was plotted using all time points and using Prism1 software (GraphPad Software). Atrazine exposure resulted in a statistically significant decrease in the LH surge at the 1600 and 1800 hour time points ( ↓62% {p<0.001} and 57% {p<0.01}, respective ly). Non

- statistically significant but substantial decreases in LH circulating levels were also noted at the 1300 and 2000 hours ( ↓43 and 51%, respectively). The LH peak was reduced by 51% (p<0.001) while the AUC was decreased by 53% (p<0.01) after a 4

- da y exposure to atrazine at 100 mg/kg/day. Statistically significant (p<0.001) increases in corticosterone levels were only noted at the 1300 hour ( ↑102%). A non

- statistically significant increase in corticosterone was also noted at the 2000 time point ( ↑2 2%). At all other time points, corticosterone levels in the treatment group were lower than in control ( ↓7

- 36%). Though not statistically significant, the corticosterone peak for the atrazine treated group was slightly higher than for control ( ↑16%) whil e the corticosterone AUC was comparable between both groups.

The study author concluded that atrazine exposure at a dose level of 100 mg/kg/day for 4 consecutive days resulted in an attenuated LH surge and an increase in corticosterone levels. Clinical signs observed after this treatment were not severe (urogenital, hindlinmb, and mouth staining) and the body weight decreases were not substantial ( ≈6%) and of questionable toxicological significance.

Reviewer’s comments: This is a well

- conducted study that partially corroborates previous findings reported by Cooper et al. (2007). The LH surge

– though not completely abrogated by atrazine treatment at 100 mg/kg/day

– was significantly dampened. However, unlike previous reports, this study suggests that corticosterone levels do not remain elevated after atrazine exposure but rather return to baseline (or even lower) shortly after.Page 17 of 210 A.2.4 Coder P. (2011e). Atrazine: An Oral (Gavage) Study of the Effects of Atrazine on the Estrous Cycle and the Estrogen

- In duced Luteinizing Hormone Surge in Female Rats. Final Report. Replaces MRID 48180103) Unpublished data from Syngenta Crop Protection, Inc. Study completion date: February 4, 2011. Report Number: WIL

639060. MRID 48381001

The purpose of this study was to assess the effects of atrazine on sexual maturation (as indicated by the occurrence of vaginal opening), the estrous cycle, and the estrogen

induced luteinizing hormone (LH) surge in F 1 female Sprague

- Dawley [Crl:CD(SD)] rats (approximately 13

- 14 weeks of age on gestation day 0) following oral gavage dosing to two separate Cohorts (I and II) of animals at atrazine doses of 0, 6.5, 25 or 50 mg/kg/day. In Cohort I, F 1 females were exposed to atrazine from conception through sexual maturation (vaginal openin g) or through PND 133. In Cohort II, naïve (untreated) F1 females were exposed to atrazine beginning at weaning (PND 21) through sexual maturation or for 14 days in adulthood (PND 121

- 133).

Study Design **:** Atrazine was administered daily via oral gavage to Crl:CD(SD) rats (F 0 females and/or their F 1 female offspring) at dose levels of 0, 6.5, 25 or 50 mg/kg/day. The study was divided into 2 cohorts (I and II) each with 4 dose groups of 25 bred F 0 female rats (approximately 13

- 14 weeks of age on GD 0). In ea ch cohort, the F 1 female offspring were further divided into 3 subsets. Each subset consisted of 15

- 25 F 1 females/group. The study design is shown in the following table and figure (taken from pages 23

- 24 of Syngenta study report).Page 18 of 210 GD = Gestation Day LD = Lactation Day PND = Postnatal Day VO = Day of vaginal opening OVX = Day of ovariectomy B/N = Day of blood collection and necropsy E = Beginning of 3

- week estrous evaluation period

Cohort I : Mated female rats (25/group) (F 0 generation) were administered atrazine (0, 6.5, 25 or 50 mg/kg/day) by oral gavage from gestation day 0 (GD 0) through lactation day 20 (LD 20) and continued administration of the F 1 offspring (F 1 generation) from weaning (PND 21) until 5 days after vaginal opening for a t otal of 15

- 26 doses (Subset A). The F 1 females in Subsets B and C of cohort I were administered atrazine following weaning (beginning on PND 21) until PND 133, which was the day euthanasia for Subset B. Subset C had a 3

- week post

- treatment recovery period . Subsets B and C received a total of 113 doses.

Cohort II: Naïve (untreated) F 0 females were utilized exclusively to generate naïve F1 offspring. Naïve F1 females were assigned to subsets D, E, or F. The F 1 females in Subset D were administered atrazi ne (0, 6.5, 25 or 50 mg/kg/day) following weaning (beginning on PND 21) and continuing until euthanasia (5 days following attainment of vaginal patency) for a total of 15

- 26 doses. The F 1 females in Subsets E and F of cohort II were administered atrazine from PND 120

- 133 (for 2 weeks prior to the scheduled LH surge evaluation and euthanasia (Subset E) or initiation of a 3

- week post

- treatment recovery period (Subset F). Subsets E and F received a total of 14 doses. AnimalsPage 19 of 210 Female Sprague

- Dawley [Crl:CD(S D)] rats (70 days old) were received from Charles River Laboratories (Raleigh, NC) and housed singly in a room maintained at 71 **±** 5oF (22 **±** 3oC) with a 14:10 h light:dark schedule (lights on at 0500 hours: lights out at 1900 hours). Following acclimation, th e rats were paired for mating in the home cage of the male. Following positive evidence of mating, females were individually housed in plastic maternity cages with nesting material. The F 0 females were housed in these cages until lactation day 21, the sche duled day of euthanasia. Following weaning, F 1 offspring were housed together by litter in plastic maternity cages with nesting material until PND 28. At PND 28, F1 offspring were housed individually in suspended wire

- mesh cages until euthanasia. LLC Certi fied Rodent LabDiet®5002 and water were provided ad libitum

. Dosing: All animals were dosed at approximately the same time each day. F 1 females in Subsets B and C, which received a total of 113 doses, were given daily doses at approximately 6

- 9 hours post

- lights (1100 hours to 1400 hours). All other animals on study (F 0 females in Cohort I and F 1 females in Subsets A, D,E,F) which received 14

- 43 doses of atrazine, were given daily doses at approximately 7

- 8:45 hours post

- lights on (1200 hours to 1345 hours ).

Dosing Solutions and Procedures

Atrazine (purity 98.8%) was received from Syngenta Crop Protection, Inc (Greensboro, NC). All dosing was administered by oral gavage in a suspension of 1% methylcellulose/deionized water in a volume of 5.0 mL dosing so lution/kg body weight. Control animals received 1% methylcellulose vehicle (5.0 mL/kg) only.

Dose levels were selected based on the results from previous studies. The highest dose level of 50 mg/kg/day was selected because it was shown by Foradori et al 2 009 to be a dose that had an effect on the LH surge in female Wistar rats and higher than the effective dose of 30 mg/kg/day seen in female SD rats fed atrazine for 6 months in a study by Morseth 1986. The low dose of 6.5 mg/kg/day was selected to be sligh tly higher than the previously identified NOAEL for atrazine on LH

- dependent effects in male rats (Stoker et al. 2000).

To eliminate endogenous estradiol, ovariectomies were performed on 1

- 2 F 1 female pups/ litter (maximum of 20 females/group/subset) at t he following time points: the second day following the day of attainment of vaginal patency (Subsets A and D), PND 130 (Subsets B and E) and following a 3

- week post

- treatment period (PND 151; Subset C and F). At the time of ovariectomy, a femoral vein cath eter attached to an SAI Quick Connect **** and a subcutaneous silastic capsule containing 4 mg/mL of estradiol benzoate in sesame oil were implanted in each female. Three days following ovariectomy, blood samples for hormone (LH and corticosterone) evaluated b y radioimmunoassay were collected from all surviving females (that underwent surgery) via the surgically implanted femoral vein access port at 6, 9, 11, 13, 15 and 18 hours post

- lights on (lights were turned on at 0500 hours and turned off at 0900 daily). Following the 18

- hour post

- lights on blood sample collection, females were euthanizedPage 20 of 210 and terminal blood samples for plasma analysis of atrazine (and metabolites) were collected from the vena cava. Each female was subjected to gross necropsy and examined for the absence of ovaries, the presence of the estradiol capsule and the presence and placement of the femoral vein catheter.

Results:

Cohort I:

Gestational and lactational exposure to atrazine in F 0 mated females resulted in reduced F 1 postnatal survival, and lower pre

- weaning pup body weight and body weight gains at 50 mg/kg/day. Continued administration of atrazine to F 1 females following weaning resulted in lower post

- weaning body weights and body weight gains at 50 mg/kg/day (Subset s A, B and C).

A 1.4

- to 2.2

- day delay in the age of attainment of vaginal patency was noted in the 50 mg/kg/day group F 1 females in Subsets A, B, C. When these data were combined there was a 1.6

- day delay (p<0.01) in the age of attainment of vaginal pa tency noted at 50 mg/kg/day. The mean body weight on the day of the attainment in the 50 mg/kg/day was similar to the controls. At 6.5 and 25 mg/kg/day there were no effects on the ages of attainment of vaginal patency or body weights at attainment for F 1 females in Subsets A, B or C of Cohort 1. The combined data are presented below.

There was also no dose

- related effect on the mean estrous cycle length for Subsets B and C.

Cohort II

Administration of atrazine from PND 120

- 133 to F1 females in Subsets E and F resulted in lower mean body weight gain and/or losses during week 1 of treatment at 25 mg/kg/day and throughout the 2

- week treatment at 50 mg/kg/day.

Atrazine administration at 6.5, 25 and 50 mg/kg/day had no effect on the attainment of

va ginal patency for F 1 females in Subsets D, E, and F or on the mean estrous cycle length for F 1 females in Subsets E and F. In Subsets D and E, vaginal patency was attained prior to the beginning of atrazine treatment on PND 120.

Plasma Hormone Analysis: An LH surge was detected in all treatment groups (0, 6.5, 25 and 50 mg/kg/day) for Cohort I (Subsets A, B, C) and Cohort II (subsets D, E, F).Page 21 of 210 Also for both cohorts and corresponding subsets there were no significant differences in either the peak heig ht of the LH surge or the area under the curve for the LH surge in any of the treatment groups. These results are unusual in that they contradict the results of other studies by Foradori et al, Morseth et al. and Cooper et al. showing that doses of 50 mg/k g/day and lower result in a statistically significant decrease in LH surge.

Corticosterone: There were no significant differences in plasma CORT levels, peak CORT levels, or AUC for CORT in any treatment group (cohorts I and II and their subsets), compar ed to controls. The study authors, however, stated that the timing of blood collection was not ideal for elucidating an effect on corticosterone. [Three days following ovariectomy, blood samples for corticosterone were collected from all surviving females via the surgically implanted femoral vein access port at 6, 9, 11, 13, 15 and 18 hours post

- lights on (lights were turned on at 0500 hours and turned off at 0900 daily).]

Concentrations of atrazine and metabolites in Rat plasma: Plasma concentrations of atrazine and its chlorometabolites 10.5 to 12 hours after dose administration indicated that little or no atrazine remained in the plasma. The chlorometabolites (DIA, DEA and DACT) were detectable at all dose levels. The concentrations of DACT were great er than DEA and DIA.

**Reviewer’s Comments:** The impetus of this study was to see whether or not pre

- or perinatal exposure to atrazine would shift the dose

- response curve to the left. The results seem to indicate that this is not the case. Gestational an d lactational exposure does not seem to be affecting cycling. The results showed a delay in the age of attainment of vaginal patency in the 50 mg/kg/day dose group.

Technically, this was a well conducted study. No effect on LH surge was seen at 6.5, 25 mg/kg/day, or 50 mg/kg/day. The lack of a specific effect on LH surge at ≤50 mg/kg/day in OVX

- estrogen primed animals in this study contradicts the results of other studies by Foradori et al.2009, Cooper et al 200, and Morseth et al 1996. Five daily dose s of 50 or 100 mg/kg/day atrazine administered to ~13 week old Wistar rats caused a significant suppression of the LH surge (Foradori et al. 2009). Four daily doses of atrazine suppressed LH secretion at 6.25 mg/kg/day in Long

- Evans female rats (Cooper et al. 2007). Using a ovariectomized, estrogen primed model, Morseth et al. 1996 reported a significantly lower dose (3.65 mg/kg/day) of atrazine was required to decrease the LH surge in females treated for a period of 6 months prior to measuring the hormon e. No changes in CORT levels were noted at any dose, however the study authors stated that their timing of blood collection was not ideal for elucidating an effect on CORT.

A.2.5 Cooper, R., Buckalew, A., Fraites, M., Goldman, J., Laws, S., Narotsky, M., Stoker, T. 2010. Evaluating the Effect of the Chlorotriazine Herbicide Atrazine on the Amplitude of the Pre

- ovulatory LH surge in the Long

- Evans Rat. EPA Internal Report.Page 22 of 210 In this study, the authors continued the work reported in Cooper et al. 2007 trying to identify a NOAEL for attenuation of the LH surge in the LE rats after 4 days of exposure to atrazine. Previous research indicated that a dose as low as 6.25 mg/kg/day for 4 consecutive days resulted in attenuation of the LH surge. Thus, t he work reported herein includes lower doses to try to establish a NOAEL.

Regularly cycling females were exposed to atrazine (purity: 97.1% a.i.) starting on the day of vaginal estrous until the day after proestrous (4 consecutive days) to 0, 25, or 75 mg /kg/day [Block 1]; 0, 6.25, 12.5, 25 mg/kg/day [Block 2] or 0, 1.56, 3.12, 6.25 mg/kg/day [Block 3]. The number of animals used in the analysis varied because in order to be included they had to meet 3 criteria at the time of the terminal sacrifice: (i) h ave a proestrous vaginal smear, (ii) increased uterine weight, (iii) increased progesterone concentration. To assess for the spontaneous ovulatory LH surge animals were decapitated at 1200, 1400, 1600, 1800, or 2000 hrs. Radioimmunoassays were used to me asure serum LH and progesterone levels.

Uterine weights, progesterone levels, and the onset of the LH surge ( i.e., timing) were unaffected by atrazine treatment in females reaching proestrous. The magnitude of the LH surge, however, was statistically sig nificantly decreased at 3.12 mg/kg/day (analysis of the data for the entire day) or 6.25 mg/kg/day (analysis for the time of peak LH surge; 1800 hr) ( ↓ ≈27% and ≈46%%, respectively).

Reviewer Comments: This is a well

- conducted study that provides valuabl e information for both hazard characterization and risk assessment purposes. Serum progesterone levels, uterine weight, and timing of LH surge were unaffected for animals reaching proestrous but the magnitude of the LH surge was dampened at doses ≥ 3.12

m g/kg/day. A NOAEL of 1.56 mg/kg/day has been identified for the attenuation of the LH surge. The reviewer considers selection of this lower dose as a NOAEL appropriate since it takes into consideration the totality of the data rather than concentrating i n one specific time point (1800 hrs the time of the afternoon LH surge) which would have resulted in a NOAEL of 3.12 mg/kg/day. These data are amenable to benchmark dose analysis to more clearly describe the dose

- response curve for the attenuation of the LH surge.

A.2.6 Dooley, G.P., Reardon, K.F., Prenni, J.E., Tjalkens, R.B., Legare, M.E., Foradori, C.D., Tessari, J.E., Hanneman, W.H. (2008). Proteomic analysis of diaminochlorotriazine adducts in Wistar rat pituitary glands and LβT2 rat pituitary cells . Chem Res Toxicol 21:844

- 851.

In a protein adduct study using immunohistochemistry and Western blot techniques, ovariectomized Wistar female rats (4/group) were administered oral gavage doses of 0 or 200 mg/kg atrazine (97.9%) in carboxymethylcellulose and sacrificed on postexposure da y 5. Pituitary glands were harvested, and either frozen in liquid nitrogen or fixed with formalin. Formalin

- fixed tissues were embedded in paraffin, sectioned, processed for immunohistochemistry and incubated with a 1:100, 000 dilution of commercial DACT adduct antibody overnight at 4° C. Prepared slides were visualizedPage 23 of 210 with a Vectastain Universal ABC kit, counterstained with hematoxylin and examined for DACT

- protein adducts. For the two

- dimensional electrophoresis, pituitary glands were frozen until use

. A similar approach was taken to prepare the LβT2 gonadotrope cells for DACT

- protein adducts analysis and for the two

- dimensional electrophoretic analysis. Cell cultures of LβT2, grown in culture medium to 80% confluency were plated at a density of 400, 000 cells on to cover slides and allowed a 48

- hour attachment period. Attached cells were exposed to 300 μM DACT, culture medium or the solvent (dimethyl sulfoxide, DMSO) for 24 hours. Treated cells on slides were rinsed, fixed in methanol, mixed with Tr iton X, blocked with phosphate buffer, incubated with the DACT adduct antibody, washed and reincubated with commercial goat antirabbit antibody labeled with the fluorescent dye, Alexa Fluor 488, and examined for DACT

- protein adducts. Under similar culture conditions, LβT2 cells were grown to 80% confluency, exposed to 300 μM DACT, culture medium or DMSO for 24 hours and frozen in liquid nitrogen until use.

For the two

- dimensional electrophoresis, either the frozen pituitary glands or LβT2 cells were tha wed, pooled, sonicated, and centrifuged. Supernatants were removed; proteins were precipitated with a commercial kit, and dissolved by sonication in 400 μL of a rehydration buffer. Samples of the tissue or cellular proteins were plated onto trays contain ing gel and subjected to electrophoresis. At the end of the procedure, the two dimensional gels were either stained with Sypro Ruby (for protein detection) or subjected to Western blotting for detection of the DACT

- adducted proteins. Protein spots on th e Western blots were matched to spots on the Sypro Ruby

- stained gels. Matched spots from pituitary tissue samples were excised from the Sypro Ruby

- stained gels, digested, rinsed and proteins were identified through MALDI

- TOF/TOF MS. Matched spots from th e cell cultures were also excised from the Sypro Ruby

- stained gels, digested, rinsed and processed for MS. Proteins detected in DACT

- exposed LβT2 cells were identified using Sequest search of the MS/MS data. For both sample types, MS spectra

- identified pr oteins were searched against the National Center for Bioinformation (NCBInr) protein databases using the taxonomy filter Rattus

. DACT formed 19 spots and covalent adducts with 8 unique proteins in the rat pituitary and 21 spots and covalent addu cts with 19 unique proteins in the LβT2 cells. The investigators reported that the proteins found in both sample types were: proteosome activator complex subunit 1 (involved in proteosomal degradation but effect on protein stability or function is not kn own); ubiquitin carboxyl

- terminal hydrolase isozyme L1 (involved in ubiquitin

- tagged protein proteosomal system degradation which is critical to cell growth, differentiation, development and transcription); tropomyosin (this protein binds to actin, one of the major components of the cytoskeleton and acts as a mediator of internal cell motility); and ERp57 (an oxidoreductase found in the endoplasmic reticulum of cells; exact function is not completely understood). In addition, RNA

binding proteins containin g active

- site or solvent

- exposed cysteine residues were also detected in both sample types.Page 24 of 210 Based on the results of this study in combination of the other Dooley studies (in PK section below) along with other studies, the authors speculate that “Because DACT is capable of forming covalent protein adducts and brain tissue is exposed to large quantities of DACT following atrazine exposure, specific modifications of proteins within the pituitary could cause aberrant function, leading to the observed suppres sion of the LH surge.”

Reviewer’s Comments: Adduct formation may be important in the regulation of pituitary function since adduct formation can be seen in the pituitary glands of rats exposed to either atrazine or DACT as well as in L β T2 Rat Pituitary C ells . I n vivo exposure to 200 mg/kg atrazine resulted in 8 DACT

- protein adducts. Similarly, L β T2 rat pituitary cells exposed in vitro to 300 μM DACT led to adduct formation between DACT and 19 proteins. This study was not designed to study a potential do se

- response in terms of adduct formation nor does it provide any data pertaining to the functional outcomes of these adducts. Nevertheless, it is noteworthy that these data entail exposure to extremely high levels of atrazine and/or metabolites that far e xceed the potential environmental exposures.

A.2.7 Fraites, M.J.P., Cooper, R.L., Buckalew, A., Jayaraman, S., Mills, L., and Laws, S.C. (2009). Characterization of the Hypothalamic

- Pituitary

- Adrenal Axis Response to Atrazine and Metabolites in the Female Rat. Tox. Sci. 112(1):88

- 99.

In this article the authors reported the results of 3 studies. The objective was to investigate if atrazine and/or two of its metabolites

– desethylatrazine (DEA) and diamino

- s

- chlorotriazine (DACT)

– affected the hypothala mic

- pituitary adrenal (HPA) axis in females by monitoring the levels of circulating adrenocorticotropic hormone (ACTH), corticosterone, and progesterone. Previously, it had been demonstrated that these hormones were affected in males after a single exposu re to atrazine at a dose level of 100 mg/kg/day. In addition, studies were also conducted to determine if the effects seen in these hormones were similar to those elicited by restraint stress or if they were secondary to gastrointestinal discomfort.

Stud y 1: Oral Chloroatrazine Exposure and Restraint Stress

Single exposure

Four to nine regularly cycling Long

- Evans female rats were treated by gavage with a single dose of either1% methyl cellulose (vehicle), 75 mg/kg atrazine (purity 97%), 60.2 mg/kg DI A, or 50.6 mg/kg DACT (atrazine equimolar doses) on the morning of proestrus. Precisely 15 minutes after treatment, the animals were sacrificed and their trunk blood collected to assay for ACTH, corticosterone, and progesterone. At necropsy, the pituitar y and uteri were collected and weighed. In addition to examining the impact of chloroatrazine exposure on hormone levels, the investigators also evaluated the impact of restraining non

- treated animals for 5 minutes on stress hormone levels as well as the pituitary and uterine weights.Page 25 of 210 With the exception of a statistically significant 33% uterine weight decrease after DACT exposure, neither the 5 minute restraint nor treatment with a single dose of atrazine or DIA affected body weight, pituitary or uterine weights. In contrast, a single dose of atrazine or DIA resulted in a statistically significant increase in ACTH, corticosterone, and progesterone within 15 minutes of exposure. Interestingly, DACT though the most prevalent of the metabolites present in the plasma after atrazine exposure, had little or no impact on any of the stress hormone levels.

Restraining the animals for 5 minutes and sacrificing them precisely 15 minutes after resulted in a statistically significant increase in ACTH, progesterone, and corticosterone levels. While the magnitude of the progesterone and corticosterone response was similar to that elicited by atrazine or DIA treatment, ACTH increases after restraint stress were markedly lower than the responses following compound expos ure.

Repeated exposure

Nine to 14 regularly cycling females were treated by gavage with vehicle, 12.5 or 75 mg/kg/day atrazine, 10 or 60.2 mg/kg/day DIA, or 8.4 or 50.6 mg/kg/day DACT {atrazine equimolar doses] for 4 consecutive days starting on the morning of vaginal estrous an d ending on the morning of proestrous. As was done during the single exposure study, animals were sacrificed precisely 15 minutes after the last administration of the compound and their trunk blood collected to assay for ACTH, corticosterone, and progeste rone. At necropsy, the pituitary gland and the uteri were collected and weighed. In addition to examining the impact of chloroatrazine exposure on hormone levels, the investigators also evaluated the impact of restraining non

- treated animals for 5 minute s on four consecutive days.

Body weight and uterine and pituitary weights were unaffected by compound administration though a statistically significant 20% increase in both absolute and relative uterine weights were observed after the animals were restr ained for 5 minutes. Consistent with what was observed after a single exposure, repeated atrazine and DIA exposures led to increases in ACTH, corticosterone, and progesterone. All of these increases were statistically significant except for the ACTH incr ease seen after treatment with 12.5 mg/kg/day of atrazine. Again, DACT appeared to have little or no impact on stress hormone levels. Interestingly, though statistically significant, the ACTH response after repeated exposure was markedly attenuated from the response after an acute exposure while the magnitude of the response for both corticosterone and progesterone were similar after acute or repeated exposures.

Atrazine, DEA, DIA, and DACT plasma concentrations were obtained 15 minutes after the last tr eatment under both exposure paradigms. After 15 minutes, the plasma concentrations were ranked such that atrazine<DEA<<DIA<DACT. After 4 consecutive exposures to 75 mg/kg/day atrazine, DACT plasma concentrations were ≈400

- fold higher than the parent comp ound and 7

- fold higher than DIA.

Study 2: VagotomizedPage 26 of 210 The objective of this study was to determine if the increase in stress hormones observed after atrazine, atrazine

- metabolites treatment were related to irritation of the gastrointestinal tract rather than a direct effect on the HPA axis.

Ten vagotomized rats and 10 intact rats were used in this experiment. Four to 5 of the vagotomized or intact rats received either 100 mg/kg atrazine or vehicle (1% methylcellulose) by gavage. The animals were sacrificed precisely 15 minutes after treatment and the trunk blood collected for hormone analysis. Atrazine treatment resulted in an increase in ACTH, corticosterone, and progesterone levels in both vagotomized and intact animals. For ACTH and progeste rone, the response in vagotomized animals was substantially more robust than in intact animals. Corticosterone levels in both intact and vagotomized animals were also increased after atrazine treatment but the difference was not as marked. Nonetheless, t hese findings suggest that the stress hormone release noted after atrazine or atrazine

- metabolite administration is not the outcome of GI discomfort.

Study 3: Intravenous DIA Exposure

This study was also conducted to evaluate if the stress hormone releas e observed shortly after atrazine or atrazine

- metabolites treatment was due to GI discomfort. In this study, rats with an indwelling jugular vein catheter were treated with either vehicle or 1 ml of 7.8 mM DIA (thus bypassing the GI tract). Blood was dra wn 15 minutes after treatment and assayed for ACTH and corticosterone levels. Both ACTH and corticosterone levels were statistically significantly increased 15 minutes after DIA administration relative to animals exposed to vehicle; suggesting that the re lease of stress hormones was not due to GI discomfort but rather a direct effect of atrazine or its metabolites on the HPA axis.

The authors of these studies concluded that, in addition to the hypothalamic

- pituitary

gonadal (HPG) axis, atrazine treatment also affects the HPA axis as evidenced by the increase in levels of circulating ACTH, corticosterone, and progesterone. Given that activation of the HPA axis has been shown to impact reproductive outcomes (likely through interactions of corticotropin

- rele asing factors (CRF) and gonadotropin release hormone (GnRH) neurons), atrazine’s effect on the HPA axis may represent a precursor event to the attenuation of the LH surge observed after atrazine exposure.

Reviewer’s Comment: The findings in these series of studies is consistent with observations in female mice where atrazine exposure to 500 mg/kd led to an increase in corticosterone levels. Similar to what was reported in this study, male rats exposed to a single oral dose to atrazine, DIA, or DEA exper ienced an increase in ACTH, corticosterone and progesterone. Interestingly, DACT appeared to have little or no impact on the HPA axis although it is the most prevalent of the chloroatrazine metabolites. Thus, it appears that atrazine and/or the intermedi ate metabolites (DEA or DIA) are critical in the activation of the HPA axis. ACTH release, however, is attenuatedPage 27 of 210 after repeated exposures to atrazine or DIA yet corticosterone levels remained markedly elevated and were comparable after a single or multip le exposures.

This study is relevant for the risk assessment and supports the MOA for atrazine. These observations strongly suggest that the atrazine

- induced endocrine response is a direct, chemical

- induced change in the hypothalamic

- pituitary

- adrenal tis sue and not the result of a non

- specific stress response.

A.2.8 Gojmerac, T., Pleadin, J., Zuric, M., Rajkovic

- Janje, R., and Korsic, M., 2004. Serum Luteinizing Hormone Response to Administration of Gonadotropin

Releasing Hormone to Atrazine

- Treated G ilts. Vet Human Toxicol. 46(5): 245

- 247.

This study looked at porcine serum luteinizing hormone levels in response to administration of gonadotropin

- releasing hormone to atrazine

- treated pigs.

From the beginning of estrous (days 0 and 21), 10 female pigs (ages 7

- 8 months) were given 1 mg atrazine/kg bw (99% purity) in the feed daily for 20 days of the estrous cycle. There were 5 female control pigs. One hour following the last treatment on day 20 (when the first signs of estrous were observed), blood samples were collected (min 0) from control and atrazine

- treated groups. Five atrazine

- treated pigs were immediately given an i.v. dose of 100 μg GnRH. Blood sample for porcine LH (pLH) determination were collected from the atrazine

- exposed and the GnRH

- injected atrazine

- treated groups at 20, 30, 40 and 90 minutes. Serum concentrations of pLH were determined by radioimmunoassay.

The experiment was preceded by another which measured pLH concentration in peripheral blood samples from 3 intact pigs during the estrous cycle. Serum concentrations of pLH were determined in samples collected from the onset of estrous (days 0 and 21) between 8 and 9 am, and then twice daily at 3

- hour intervals, and on days 1,2, 4, 6, 8,10, 12, 14,16,18,19. and 20 of the estrous cycle.

The pLH concentration from 3 normally cycling (intact) mature female pigs measured at 3 daily 3

- hr intervals during the estrous cycle was increased from 2.6 to 3.9 ng/ml on days 0 and 21, which indicated the onset of estrous. The mean serum pLH

co ncentration in control animals on day 20 of the estrous cycle correlated with the levels in the 3 normally cycling animal on the same day. Repeated exposure of female pigs to atrazine resulted in significant reduction (p<0.001) of serum pLH on day 20 of t he estrous cycle (0.57 ±0.05 ng/ml) compared to control animals (2.24±0.20 ng/ml), which is consistent with other studies showing significantly reduced estrogen

- induced LH response following atrazine administration in female rats. The single iv GnRH admini stration to atrazine

- treated female pigs resulted in significant increase in pLH 20 and 30 minutes after GnRH administration, indicating that attenuation of GnRH release in atrazine

- treated animals was responsible for the suppression of serum pLH secretion

. Reviewer’s Comments: This study is relevant to risk assessment in that it supports the neuroendocrine MOA identified for atrazine. By demonstrating that suppression of thePage 28 of 210 LH secretion can be abrogated by providing an exogenous source of GnRH, the stud y authors have clearly demonstrated the relationship between disruption of GnRH's pulsatile release and attenuation of the LH surge.

A.2.9 Juliani, C., Silva

- Zacarin, E., Santos, D. and Boer, P. 2008. Effects of Atrazine on Female Wistar Rats: Morphologic al Alterations in Ovarian Follicles and Immunocytochemical Labeling of 90 kDa Heat Shock Protein. Micron. 39:607

- 616.

The purpose of this study was to characterize morphologically the effect of low and sublethal doses of atrazine on ovaries of adult fema le Wistar rats to determine the possible intrafollicular target site through which atrazine acts. In addition, they analyzed the expression of the 90 kDa heat shock protein in both ovarian follicular cells and ooctye by immunochemical labeling, to evaluate the role played by this protein on the ovary under stressed conditions, induced by atrazine exposure.

Female Wistar rats (n=5/dose group) were dosed via oral gavage with atrazine (97.1% purity) at doses of 0 (vehicle), 0.75 and 400 mg/kg/day. The suble thal dose of 400 mg/kg/day was chosen since it is two

- fold the atrazine dose responsible for disruption of the estrous cyclicity in female rats (Laws et al 2000). The dose of 0.75 mg/kg/day was chosen since it was the lowest dose of atrazine that had been tested on female rats which had not induced estrous cyclicity alterations (Cooper et al. 1996; Laws et al. 2000). The study was conducted in two blocks. In the subacute treatment block, females received 0 or 400 mg/kg/day atrazine for 14 days. In the su bchronic treatment block, the females received 0 or 0.75 mg/kg/day atrazine for 30 days. Body weights were recorded daily. At the end of the treatments, the animals were euthanized and ovaries were removed and prepared for histology and immunohistochemical analysis. Primary, preantral, and antral follicles per ovary were analyzed.

In the subacute group, there was a decrease in food consumption and animals lost ~19% of their initial body weight after 14 days, while the control group had a 17.2% increase in body weight. Clinical signs included fatigue and weakness in the treated animals, slight trembling in hind legs and presence of strong odor in urine. It should be noted that none of these data were provided in the article. In the subchronic group, there was no significant decrease in food consumption in the 0.75 mg/kg/day dose group. There was an increased body weight (8.5%) in the dosed animals after 30 days. This was lower than the weight gain in the control group (18%). Clinical signs included fatigue and weakness.

In the subchronic treatment group (0.75 mg/kg/day for 30 days, rare multioocytic follicles were observed in the treated group. In the subacute treatment group (400 mg/kg/day for 14 days), atrazine induced oocyte toxicity as evidenced by the presence of preantral follicles with disorganized granulosa cells and/or a degenerating oocyte with discontinuous zona pellucida, which were not observed in the control group. Ovotoxicity was also observed in antral follicles in varied degrees. In some a tretic antral follicles,Page 29 of 210 where high intensity of apoptosis was observed in the granulosa cells, the oocyte showed nucleolus fragmentation and irregularities of the Zona Pellucida.

Low doses of atrazine induced an increased expression of HSP90 in follicula r granulosa cells in comparison to the control group. However, the positive reaction product, indicative of the HSP90 expression, was not found in follicular granulosa cells of ovaries collected from rats exposed to subacute treatments.

Reviewer’s Comment s: It should be noted that in this study no quantification of findings was attempted. Most results were presented in the form of photomicrographs, thus it is difficult to assess the relevance of the findings since most of the assessments were subjective (e .g. visual inspection of color change in cells.) This study, therefore, is not very useful for risk assessment.Page 30 of 210 A.2.10 Laws, S., Farrell, J., Stoker, T. and Cooper, R. (2003) Pubertal Development in Female Wistar Rats following Exposure to Propazine and Atrazine Biotransformation By

- Products, Diamino

- S

- Chlorotriazine and Hydroxyatrazine. Toxicological Sciences 76:190

- 200.

The purpose of this study was to evaluate the effects of two atrazine by

- products, diamino

- s

- chlorotriazine (DACT) and hydroxyatr azine (OH

- ATR), and a structurally similar chlorotriazine, propazine (PRO) on female pubertal development. Previous studies by Laws et al. have shown that atrazine delays the onset of pubertal development in female rats.

Female Wistar rats (n=15/treatment group) were gavaged from postnatal days (PNDs) 22 through PND 41 with DACT (96.8% purity) (16.7, 33.8, 67.5, 135 mg/kg), OH

- ATR (97.1% purity) (22.8, 45.7, 91.5, 183 mg/kg) or PRO (99.8% purity) (13, 26.7, 53, 106.7, 213 mg/kg). The dose range for each chemical was selected as the molar equivalent of atrazine (12.5, 25, 50, 100, 200 mg/kg). The females were monitored daily for vaginal opening (VO) and killed on PND 41. The age at complete vaginal opening was recorded. Body weights were re corded daily. Tissue weights for each animal were recorded for liver, kidney, adrenals, ovaries, uterus, and pituitary. Histological evaluations were performed on the thyroid, uterus, and ovaries. [Note: The Protocol for the Assessment of Pubertal Develo pment and Thyroid Function in the Female Rat (Goldman et al. 200) was used for these studies. Since this protocol is undergoing validation by US EPA, thyroid hormones were evaluated in these studies even though no previous data suggested that chlorotriazi nes would affect thyroid function.]

DACT (33.8, 67.5, and 135 mg/kg) significantly (p<0.05) delayed the age at vaginal opening by 3.2, 4.8, and 7.6 days, respectively, compared to controls (33.1± 0.4 (SE) days of age). Four of 15 animals in the highest D ACT dose group did not attain vaginal opening by necropsy at PNDs 41

- 42. Body weight at the age of VO was also significantly increased in the three highest dose groups of DACT (13

- 19%). At necropsy the body weight in the high dose DACT animals remained significantly lower (10.4%) than controls. PRO (106.7 and 213 mg/kg) also significantly delayed the age of VO by 3.6

- 4 days. A significant increase in body weight at VO was seen in females receiving the highest dose of PRO. Doses of OH

- ATR (up to 200 mg/k g) did not cause a significant delay in the age of VO.

Significant reductions in kidney, pituitary, and adrenal weights were observed following exposure to the highest dose of DACT. No significant differences in tissue weights were seen in any of the OH

- ATR animals. Pituitary weights in the two highest dose groups of PRO (106.7 and 213 mg/kg) were significantly decreased compared to controls.

No significant dose related effects were seen for serum T3 or TSH following exposure to any of the test chemical s. An absence of corpora lutea was noted in 4 animals in the highest DACT treatment group that did not attain VO prior to necropsy. No treatment

related microscopic changes were seen in any animals in the PRO or OH

- ATR groups.Page 31 of 210 This study concluded that oral exposure to DACT and PRO from PNDs 22

- 41 delays the age of vaginal opening in Wistar rats in a dose

- dependent manner. The no effect level (NOEL) for DACT (16.7 mg/kg) was identical to the equimolar NOEL for atrazine (25 mg/kg). The LOEL for delay in vaginal opening for DACT (33.8 mg/kg) was equimolar to that reported for atrazine (50 mg/kg). The LOEL for PRO was 2

- fold higher that that for atrazine (100 mg/kg vs 50 mg/kg). OH

- ATR appeared to be less potent and did not alter the onset of puberty at dos es equimolar to 200 mg/kg ATR.

Reviewer’s Comments: It appears that post

- natal exposure to DACT results in a delay in sexual maturation. These delays do not appear to be secondary to low body weight since body weight at time of VO was significantly higher for the delayed pups. Given that atrazine is quickly metabolized in the body, it appears that DACT is driving the delays in puberty onset. The DACT NOAEL for sexual maturation reported by Laws et al. (25 mg/kg/day in atrazine molar equivalents) was highe r than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently used as the point of departure in the Agency’s risk assessment.

A.2.11 McMullin, T.S., Andersen, M.E., Nagahara, A. Lund, T.D., Pak, T., Handa, R.J., Hanneman, W.H. (2004). Eviden ce that atrazine and diaminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague Dawley rats without changing estrogen receptor action. Tox. Sci. 79:278

- 286.

High oral doses of atrazine (ATRA) suppress th e estrogen

- primed luteinizing hormone (LH) surge in ovariectomized female rats. The mechanism by which ATRA mediates this action is unknown, but may be related to its anti

- estrogenic activity since LH release from the pituitary gland is largely regulated b y estrogen binding to its receptor in the hypothalamus. Since atrazine is rapidly metabolized to diaminochlorotriazine (DACT) ( i.e. , AUC for DACT is 166

- fold greater than that of ATRA at 24 hours after dosing rats with 300 mg/kg bw atrazine), it could also be the actions of this metabolite on the estrogen receptor (ER). The current study addresses this issue by dosing ovariectomized Sprague Dawley rats by oral gavage with ATRA or DACT to investigate if the effects of atrazine and/or its major metabolite dia minochlorotriazine (DACT) on the estradiol benzoate (EB)/progesterone

- induced LH surge are due to impaired ER function. As expected, ATRA suppressed the EB and progesterone

- induced LH surge in a dose dependent manner. A daily dose of 300 mg ATRA/kg bw for 5 days nearly abolished the LH surge. In contrast, a dose of 77 mg DACT/kg bw (which produces the same AUC for DACT as dosing with 300 mg/kg bw ATRA) only partially attenuated the LH surge (~ 60%). The effect of DACT on pituitary function was also examined in response to exogenously added gonadotropin

- releasing hormone (GNRH). DACT attenuated the LH surge by 47% as compared to controls, indicating a decrease in pituitary function.

Binding studies with uterine cytosolic containing ER and in vitro

- transcrib ed ER indicate that ATRA, but not DACT at the same molar equivalent concentration, decreases estradiol binding regardless of incubation time. Based on the dose dependent inhibitionPage 32 of 210 of estradiol

- binding, an inhibition binding constant (Ki) of 0.02 mM was es timated for ATRA while DACT exhibited a 10

- fold greater value.

In an effort to investigate whether ATRA alters estrogen binding to hypothalamic ER, unoccupied ER levels were evaluated in two estrogen

- responsive hypothalamic nuclei. Treatment with ATRA alo ng with estradiol benzoate did not alter the levels of unoccupied ER levels as compared to estradiol benzoate treatment alone. Moreover, ATRA failed to block estrogen

- induced progesterone mRNA expression as compared to estrogen treatment alone.

Reviewer’s Comments: In summary, the results of this study indicate that (1) although ATRA binds ER in vitro , the suppression of the LH surge in vivo may not be due to alteration of ER function in the hypothalamus by ATRA itself. This finding is not consistent with the findings of several other researchers that show that atrazine does not bind directly to the ER (Cooper et al. 2007, Albanito et al. 2008, Judson et al. (2010 ) ; (2) the difference in potency between ATRA and DACT in suppressing the LH surge may be due to pharmacokinetic differences, that is, a greater internal exposure to DACT may result from dosing with ATRA than DACT itself due faster metabolism/excretion of this species; (3) the effects of DACT in suppressing the LH surge involves at least in part de creased pituitary function.

A.2.12 Shibayama, H. Kotera, T., Shinoda, Y., Hanada, T. Kajihara, T., Ueda, M., Tamura, H., Ishibashi, S., Ymashita, Y., and Ochi, S. 2009. Collaborative Work on Evaluation of Ovarian Toxicity Two

- or Four

- week Repeated Do se Studies and Fertility Study of Atrazine in Female Rats.

Atrazine was administered via oral gavage to female (n=10/group) Sprague

- Dawley rats (>97% purity) for two or four weeks at doses of 0, 3, 30, or 300 mg/kg/day for the repeated dose toxicity stud y and at doses of 0, 3, 30, or 100 mg/kg/day for the female fertility study from two weeks before mating to Day 7 of gestation.

Repeated Dose Toxicity Studies: Dose levels of 0, 3, 30 or 300 mg/kg/day were selected for these studies based on results of previous studies that showed that treatment of female SD rats with atrazine for 21 days induced disruption of the estrous cycle at 75 mg/kg/day and inhibited foll icle and corpus luteum development at 300 mg/kg/day (Cooper et al. 1996). Treatment of ovariectomized female rats with atrazine for five days suppressed the peak level of the LH surge at 30 mg/kg/day (McMullin et al. 2004).

Five week old female SD rats w ere treated for either two or four weeks. Clinical signs and mortality were observed twice daily. Body weight/gain and food consumption were measured twice a week. Vaginal smears were taken every day and the mean estrous cycle was calculated during the dos ing period. Animals were sacrificed after 2 or 4 weeks, blood samples were taken for hematology and blood chemistry. The ovaries, uterus, and pituitary gland were weighed. These organs, as well as the vagina and mammary gland, were examined histopathologic ally.Page 33 of 210 Female Fertility Study: Dose levels of 3, 30 or 100 mg/kg/day were selected as described above. Ten week old male and female SD rats were used for the fertility study. Females were treated for two weeks as the premating period. Males were not treat ed and were only used for mating purposes. After the premating period, females were caged with untreated males on a 1:1 basis overnight for up to two weeks. Daily administration of atrazine or vehicle was continued throughout the mating period and until Da y 7 of gestation, or until the last day of the mating period if the animal did not copulate. Clinical signs and mortality were observed twice daily during the administration period and once daily during the other periods. Body weights were measured twice a week during the pre

- mating and mating periods and on Days 0

- 8, Day 11 and Day 14 of gestation period. Food consumption was measured twice a week during the premating period and on Days 1, 4, 8, 10, and 13 of gestation. The mean estrous cycle length during the pre

- mating period was calculated. Vaginal smears and vaginal plugs were checked for the presence of sperm during the mating period. On Day 14, animals were exsanguinated and the presence or absence of implantations was determined after macroscopic exa mination of the main organs. The number of corpora lutea, implantations, live embryos, and dead embryos were counted and the copulation rate, and pregnancy rate were calculated. Animals that did not copulate were necropsied 14 days after the mating period and the thoracic and intraperitoneal organs were examined macroscopically.

Results for Repeated Dose Toxicity Studies : No mortality occurred in the 2

- or 4

- week study. In the two week study, clinical signs included lacrimation seen at 30 and 300 mg/kg/da y and salivation and soiled perineal region at 300 mg/kg/day. However, no incidence for these findings was provided. In the four week study, clinical signs included lacrimation, salivation, and soiled perineal region at 30 and 300 mg/kg/day and decreased stool observed at 300 mg/kg/day. Decreased body weight/body weight gain was observed in the 300 mg/kd/ay dose groups in both the two week (11%, p<0.05/39%, p<0.01) and four week (17%/ 42%, both at p<0.01) studies.

Prolonged mean estrous cycle was observe d at the high dose in the two week study (9.3 days vs 4.2 days, control, p<0.01) and in the 4

- week study at 300 mg/kg/day (8.9 days vs 4.2 days control, p<0.01). The number of animals with irregular estrous cycles was increased (p<0.01) at 300 mg/kg/day in both the 2

- week and 4

- week studies (100% and 80% incidence vs 0

- 20% incidence in control).

Decreased absolute ovarian weights were observed at 300 mg/kg/day in the 2

- week study (19%, p<0.01) and the 4

- week study (17%, p<0.05). The uterus weight was decre ased at 300 mg/kg/day (25%) in the 4

- week study. In the two week study at the high dose, histopathological findings occurred in the ovary (loss of currently formed corpora lutea, decrease in numbers of previously formed corpora lutea, increased in large

- si zed atretic follicles, swelling of previously formed luteal cells), the uterus (atrophy), vagina (mucification and increased single cell necrosis in the vaginal epithelium) and mammary gland (lobular hyperplasia). No weight or histological changes were o bserved in the pituitary gland. In the 4

- week study at the high dose,Page 34 of 210 histopathological findings occurred in the ovary (increase in large

- sized atretic follicles (slight), swelling of previously formed luteal cells (slight)), uterus (atrophy), and mammary gland (lobular hyperplasia and increased lactation). The four week treatment group showed lower incidences of abnormal ovarian and vaginal histopathological findings then the two week treatment group. The authors noted that these results may be explained by spontaneous recovery of some animals from a disturbed to a normal estrous cycle.

Results for Female Fertility Study: No mortality occurred. Clinical signs included salivation at 30 and 100 mg/kg/day and lacrimation and decreased stools at 100 mg/kg/d ay. However, no incidence of these findings was provided. Atrazine did not show significant effects on the fertility study in rats. The copulation index, fertility index, number of corpora lutea, number of implantation sites, numbers of live and dead emb ryos, and percentage of preimplantation and postimplantation loss were not affected by atrazine treatment. It should be noted that the high standard deviations for these observations call into question the quality of the data. Copulation failure caused by prolongation of diestrus was seen in 1 animal in the 100 mg/kg/day group, which could be due to the anovulatory effect of atrazine. No histopathological findings were seen in the ovaries.

The authors concluded that the effect of atrazine on female fert ility in rats could be assessed by detailed histopathological examination of the ovaries in a two

- week repeated dose toxicity study.

Reviewer’s Comments: In summary, atrazine caused irregular estrous cycles accompanied by adverse changes in ovarian histop athology at doses of 300 mg/kg. Extended dosing for 4

- weeks did not further exacerbate the effects. In contrast, no effect on fertility was observed at a dose of 100 mg/kg. While one of the objectives of this study was to compare ovarian histology with fertility, the lack of identical doses in the repeated dose toxicity studies and the fertility studies prevented this comparison. It is noteworthy that the NOAEL for the effects reported by Shibayama et al., (30 mg/kg/day in atrazine molar equivalents) w ere higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently used as the point of departure in the Agency’s risk assessment.

A.2.13 Stoker, T.E., Zorrilla, L., Strader, L., Gibson, E., Kamel, A., Qian, Y., Podhorniak, L. 2010. The Distribution of Atrazine in the Wistar Rat Following Gestational/Lactational Exposures. **EPA Internal Report.**

In this study the authors studied the transfer of atrazine and its metabolites into the fetus or the pups after maternal exposure during gestati on (GD 18

- 20 or GD 14

- 20) or during gestation and lactation (GD14

– PND 10). Also evaluated was the potential of atrazine and/or its metabolites to cross the blood

- brain

- barrier (BBB) by measuring the amounts of compounds present in the brains of both mat ernal animals and neonates.

Time

- pregnant animals were exposed to 0, 5, or 25 mg/kg/day atrazine (97% a.i. purity) by gavage during GD 18

- 20, 14

- 20, or GD 14

- PND10. Dams sacrificed on GD20Page 35 of 210 received the compound 2 hours prior to sacrifice and the blood, b rain, adrenal and mammary glands were collected and tissues weighed. Two fetuses/sex/dam were removed from the uterus and examined for atrazine and/or metabolites content (whole body). Dams sacrificed on PND 10 were separated from their pups 3 hours befo re the time of sacrifice, treated with compound or vehicle one hour after separation from the pups, and allowed to nurse their pups for 30 minutes just before sacrifice. Dams’ tissues collected included blood, brain, adrenal, and mammary glands and solid tissues weighed. One male and one female pup per dam was perfused and the plasma, milk from stomach, adrenal glands, gonads, and brains were collected and analyzed for atrazine and/or metabolite content.

In general, the major metabolite detected in dams, fetuses, and pups was DACT although atrazine, DEA, and DIA were also detected albeit at substantially lower levels. In dams exposed solely during gestation ( i.e., GD18

- 20 or 14

- 20) , DACT accounted for ≈ 77% of the total chlorotriazines (TCT) detected in plasma, 85% of TCT in brain, 65% of TCT in mammary glands, and 60% of TCT in adrenal glands. After DACT, DIA was the most common metabolite detected with levels ranging between 11

- 20 % of the TCT detected in tissues. Atrazine per se accounted for <1% of TCTs in plasma and brain and 6 and 11% of the TCTs in mammary and adrenal glands, respectively. In fetuses, DACT accounted for ≈ 78% of the TCTs detected in the whole fetus and DIA ac counted for ≈ 18%, atrazine per se accounted for <1%).

Similarly, for dams exposed from GD14

- PND10 DACT was the most prominent metabolite. DACT accounted for ≈ 90, 95, 75, and 90% of TCTs detected in plasma, brain, adrenal, and mammary glands, respective ly. DIA, the second most common metabolite in all tissues (except the adrenals), accounted for 3

- 8% of the TCTs detected. In the adrenal glands, the parent compound (atrazine) accounted for ≈ 11% of the TCTs while in other tissues it accounted for <1%. In pups, DACT was by far the predominant metabolite detected accounting for 99, 91, 100, and 96% of TCTs in pup plasma, stomach content, adrenal glands, and brain, respectively. DIA was either not detected or detected at levels below the level of quantita tion (LOQ) in the adrenals and detected barely above the LOQ in plasma and brain at the 25 mg/kg/day dose level. In the stomach contents, however, DIA accounted for ≈6% of the TCT. Due to the small size of the gonads in the neonate, the tissues (ovaries or testes) were only analyzed for the presence of DACT. DACT was only detected in the ovaries of the offspring of dams treated at the 25 mg/kg/day dose while it was detected in the testes at both doses.

It is noteworthy that for all tissues

– except the brain

– the content of atrazine and/or its metabolites was markedly higher in dams than in their pups ( ≈80

- 97% higher). In the brain, however, the content of atrazine in the pups was comparable or ≈5

- fold higher than in the dams while the DACT and DIA con tent in the pup brain was 3

- 19% of that detected in the dams’ brain.

Table A

- 1 : Atrazine and Predominant Metabolites Tissue Levels in Dams Exposed GD14

- PND10 and their OffspringPage 36 of 210 Atrazine

dose (mg/kg/day) Tissue Content Atrazine DACT DIA

Dams Pups Dams Pups Dams Pups

Plasma (ng/ml) 0 LOQ a LOQ LOQ LOQ LOQ LOQ 5 0.9 ± 0.4 LOQ 960.5 ± 36.2 96.8 ± 4.1 (10%) b 38.3 ± 6.8 LOQ

25 9.8 ± 8.3 LOQ 2477 ± 863.6 330.7 ± 79.2 (13%) 173.8 ± 111.4 1.8 ± 0.32 (1%) Adrenal (ng/g) 0 LOQ LOQ LOQ LOQ LOQ LOQ 5 10,3 ± 4.2 2.9 ± 2 332.9 ± 151 LOQ 10.6 ± 6.8 LOQ 25 58.7 ±45.6 LOQ 424.9 ± 173.9 86 ± 50.8 (20%) 44.5 ± 28.6 LOQ

Brain (ng/g) 0 LOQ LOQ LOQ LOQ LOQ LOQ 5 1.2 ± 0.2 5.6 ± 5.4 (367%) 710.4 ± 42.5 100.1 ± 9.9 (14%) 11 ± 2.1 LOQ

25 10.6 ± 8.8 9.7 ± 10.1 (92%) 1961 ±675.8 382 ± 102.6 (19%) 55.5 ± 37.1 1.5 ± 0.36 (3%)

a Undetectable or below the level of quantitation (LOQ) b Numbers in parenthesis represent percentage of maternal content

Reviewer Comments: This is a well

- conducted study that provides valuable information on the distribution of atrazine and its metabolites into the fetus or neonates following gestational and lactational exposure to the dams. As expected given atrazine’s f ast metabolic profile, DACT was the most common metabolite found in dams, fetus, and pups in all tissues examined. In dams, DACT represented 60

- 95% of the TCT detected in the tissues tested, in fetus DACT was ≈ 78% of the TCT detected in the whole fetus

w hile in PND10 pups DACT accounted for 91

- 100% of the TCTs in the tissues tested. Overall TCT content in the fetus is at least 40% lower than in the dams (given that only selected maternal tissues were analyzed for TCT content vs whole fetus TCT content, the whole maternal TCT content is most likely higher than what was measured in this experiment). In pups, the overall TCT content for tissues examined is ≈ 80% lower than in the dams. These data demonstrate that although transfer of atrazine and/or its metabolites between the dam and her offspring occurs, the overall concentrations of these compounds reaching the tissues in fetuses and pups is appreciably lower than in the dams. Also noted is that atrazine and its metabolites can cross the BBB both in

a dults and in pups.

A.2.14 Rayner, J.L., Enoch, R.R., and Fenton, S.E. (2005) Adverse effects of prenatal exposure to atrazine during a critical period of mammary gland growth. Tox. Sci. 87(1):255

- 266.

In this study, the authors investigated the effects of prenatal atrazine exposure on mammary gland (MG) development. In particular, the objective of this study was to identify critical periods of susceptibility during which atrazine exposure may result in delays in MG development and whether the de lays previously reported were associated/secondary to other effects ( e.g., decreases in pup weight).

Two major experiments were conducted as part of this study: (i) critical period experiment

– designed to clarify the critical periods of susceptibility dur ing gestation thatPage 37 of 210 resulted in delays in MG development after atrazine exposure, and (ii) fetal weight experiment

– designed to study if decrements in maternal body weight/weight gain affected the number and weight of fetuses at the end of gestation.

Crit ical Period Experiment

**Study Design**

Two blocks consisting of 40 pregnant Long

- Evans (LE) dams per block were exposed to 0 or 50 mg/kg (2x daily for a total daily exposure of 100 mg/kg/day) atrazine. Eight dams/exposure period/ treatment were exposed for different periods during gestation. Control animals were exposed from gestation days (GD) 13

- 19 while atrazine

- treated dams were exposed during GD 13

- 15, GD 15

- 17, GD 17

- 19, or GD 13

- 19. On PND 4, the litters were culled to 10 pups/litter (6 females and 4 males, when possible). 6 Pups were weaned on PND 22 or 25, weighed and caged 2 females/cage. Female pups (2/dam, 4 dams/group/block) were sacrificed on PND4 [from culled animals], PND 22, 25, 33, 46, and 67. It should be noted that females in block 2 were retained until PND67 due to persistent effects on MG development noted in PND 46 females in the first block.

In the lactational challenge portion of the experiment, regularly cycling female pups surviving after PND 67 were bred to control males to produce an F 2 generation. On PND 4, the F 2 were standardized to10 pups/litter. Subsequently, on PND 11 these pups were separated from their dams for two hours the returned to their dams and allowed to suckle for 20 minutes before being decapitated. T runk blood was collected as well as the pituitary and mammary glands. The pituitary was weighed while the MGs were prepared for whole mount analysis. The dams were also sacrificed and their uterus examined for enumeration of implantation sites.

Twenty

- f our female pups/group were examined for vaginal opening (VO) beginning on PND 29. The day after VO, the animals were weighed. For females in Block 2, vaginal smears were collected from PND 37

- 67 to determine the impact, if any, of atrazine pre

natal expo sure on cyclicity.

Two or more female pups were terminated on PND 33, 46, or 67 by decapitation; trunk blood was collected on PND 33 and 67. On PND 33, MGs were removed and prepared for whole mount from female pups that had undergone VO. On PND 67, in a ddition to blood, the pituitary gland, ovaries, and wet uterus were removed and weighed. The MGs were also collected and prepared for whole mount.

The blood collected during the sacrifices was used to obtain serum that could be analyzed via immunoassay for levels of total testosterone, androstenedione, corticosterone, progesterone, estrone, estradiol, and prolactin.

**Results**

6 Male pups were used for another study unrelated to these investigations.Page 38 of 210 In general, exposure to 100 mg/kg/day atrazine resulted in substantial and at times statistically significant decreases ( ↓26

- 100 %) in maternal body weight gain regardless of the period of exposure. Pup mortality after in utero exposure during GD 13

- 15, 15

- 17, or 17

- 19 was increased to 33, 41, and 30%, respectively relative to control (25% mortality). Interestingly, pup weight was unaffected by treatment with atrazine until PND 67 when animals exposed in utero from GD15

- 17 or GD 13

- 19 exhibited a statistically significant (p<0.01) decrease in weight. Data not provided.

Onset of vaginal opening was slightly but statistically sig nificantly (p<0.0004) delayed ( ≈ 2 days) for pups exposed in utero during GD13

- 19 in the absence of body weight decreases. Estrous cyclicity appeared to also be unaffected by treatment with the test compound. On PNDs 4, 22, and 25 the MG area was decreas ed ( ↓32

- 60%) by treatment with 100 mg/kg/day atrazine during various gestational periods; however, length of the epithelium was not affected. When MD development was scored subjectively , the authors stated that atrazine

- exposure delayed the development o f the gland based on displays of “abundant terminal end buds in 2

- 3 sides of epithelial tree, limited lobules, and sparse branching.” These differences, however, were no longer noted during the examination of PND 33 animals though subjective scoring conti nued to consider MG development for these pups to be delayed. During the examination of PND 46 females, MG development was markedly different from control in that the MG of atrazine

- treated pups (particularly GD17

- 19 and GD 13

- 19) exhibited less dense epi thelial branching and contained many terminal end buds.

Serum hormone measurements were obtained to assess the impact

– if any

– of atrazine exposure on serum hormone levels. Hormone measures on PND 33 revealed a statistically significant increase ( ↑269% , p<0.009) in corticosterone level accompanied by a 206% increase in testosterone levels (p< 0.04). In contrast, on PND 67 none of the hormones measured or the reproductive organs (ovaries and uterus) weighed appeared to be altered by atrazine exposure.

Female pups exposed to atrazine in utero were mated to control males in an attempt to elucidate if the delays in MG development led to detrimental effects in the dams’ ability to care for their young. With the exception of one female in the GD 17

- 19 exposure group, all females mated were fertil e. Body weights and pituitary gland weights were unaffected by atrazine exposure. On PND4, the litters were standardized to 10 pups and on PND 11 the lactational challenge took place as previously described. In general, on PND 4 and 11 the offspring of dams exposed during GD 17

- 19 or 13

- 19 were smaller and weighed less ( ↓13

- 25%) than control. The MGs of female pups sacrificed on PND4 that were born to dams exposed on GD17

- 19 or 13

- 19, were taken and assessed for development. Unlike the MGs taken from c ontrol female pups, the MGs of females born to dams exposed in utero on GD 17

- 19 or 13

- 19 had few ductal buds and were undersized both during the PND 4 and PND 11 evaluations. However, when the pup body weight is used as a covariate there were no statisti cal difference between the MGs of these treated pups and control.Page 39 of 210 Fetal Weight Experiment

**Study Design**

Eight pregnant LE dams/dose were exposed by gavage to 0, 25, 50, or 100 mg/kg/day atrazine during GD 15

- 19. On GD20, the dams were weighed, sacrific ed and their litters removed. The fetuses were sexed and weighed. In addition, prolactin levels in maternal blood were measured.

**Results** Maternal body weight were statistically significantly reduced ( ↓42

- 63%) at doses ≥ 50 mg/kg/day. Neither fetal weight s nor percentage of resorbed fetuses were affected by atrazine exposure suggesting that the decreases in maternal body weight were not related to litter size (weight or number of pups). Though no statistically significant differences in the levels of prol actin were noted at any dose level, prolactin levels were decreased by 49% at the highest dose tested (100 mg/kg/day).

Conclusions

Differences seen in gestational durations were most notable from PND 15

- 17, however, at such high doses it is difficult to determine the exact critical period because the mode of action of Atrazine is unknown and also there were no developmental delays in the rat MG. There were no changes in puberty or hormones that could be associated with delayed MG development in this study and presumably because traditional endpoints were measured only, they showed no association with MG effects.

A.2.15 Davis LK, Murr AS, Best DS, Fraites MJP, Zorrilla LM, Marotsky MG, Stoker TE, Goldman JM, Cooper RL. 2011. The Effects of Prenatal Exposu re to Atrazine on Pubertal and Postnatal Reproductive Indices in the Female Rat. Reprod. Toxicol., doi:10.1016/j.reprotox.2011.04.004 In this study the authors investigated the potential impact of in utero exposure on female reproductive development as me asured by puberty onset (day of vaginal opening), mammary gland (MG) development, and estrous cyclicity. This study is the companion study to Fraites et al., (2011) in which effects of gestational exposure on male reproductive development were described. In addition, the investigators studied the transfer of atrazine and its metabolites into the fetus after maternal exposure during gestation (GD 18

- 20 or GD 14

- 20). 7 Time

- pregnant Sprague

- Dawley rats (12 dams/dose) were exposed to 0, 1, 5, 20, and 100 mg/k g/day atrazine (97% a.i. purity) by gavage once daily (s.i.d) or divided into 2 daily doses (b.i.d) during GD 14

- 21. On PND 7 pups were weighed and AGD measured. Beginning on PND 28 all female offspring were evaluated daily for vaginal opening and the bo dy weights were recorded on each day. Mammary gland development was assessed using whole mounts of the 4 th abdominal mammary gland of PND 45 old pups. This day was selected on the basis of a recommendation from a

7 The results of the atrazine distribution experiments were described in review A.2.13 in Appendix A of this document and will not be discussed further in this review

.Page 40 of 210 workshop on MG development held in 2009. 8

No effects on pup viability, pup weight, or attainment of vaginal opening (VO) are reported at do ses ≤ 20 mg/kg/day. Early postnatal pup mortality (PND0

- 4) was increased at the 100 mg/kg/day dose level in both the s.i.d. (9.7% mortality vs. 1.4 in control) and b.i.d. dosing regimen (33.6% mortality vs 0.9% control). Other effects reported at 100 mg /kg/day included decreases in pup weight after s.id. ( ↓15

- 17%) or b.i.d. (( ↓12

- 15%) exposures and delays in VO (<2 day delay). A statistically significant decrease in pup weight ( ↓10%, p<0.05) at the 5 mg/kg/day dose level (s.i.d. exposure) was not conside red to be toxicologically significant since it showed no dose

- response, had resolved by PND4, and was not observed in the b.i.d. exposure regimen. No effects on estrous cyclicity or MG development were noted at any dose level regardless of the dosing regim en. MG development was evaluated using both a morphometric approach and subjective scoring in accordance to the methodology used by Enoch et al. (2004). In the morphometric methodology, photographs of each mammary gland were evaluated using ImagePro® Softwar e linked to a camera mounted on a microscope. Measures were taken of various regions and coefficient of variation determined to assess the representativeness of the area. Branch points, terminal end buds, and the distance between the lymph nodes and the ductal field were measured using this approach. Using the procedures described by Enoch, the mammary glands were subjectively scored for their stage of development where a score of 1 represented poor MG development while a score of 4 represented normal de velopment for a PND45 rat. Finally, after all females attained vaginal patency, estrous cycles were monitored at 14 day intervals to assess cyclicity status.

Reviewer Comments: This is a well conducted study that evaluates the impact of in utero atrazine exposure on female reproductive indices including VO attainment, extrous cyclicity, and MG development. This study confirms previous findings by Rayne r et al. (2004 and 2005). Toxicologically relevant effects including decreases in pup weight, increased early postnatal mortality, and delays in VO were noted at the 100 mg/kg/day dose only. Most notably, however, is the failure to affect MG development at any dose level whether it was evaluated using a morphometric analysis or the subjective scoring described by Enoch et al. (2004). Figure 1. Quantitative analysis of mammary gland morphology in female rats prenatally exposed to atrazine on GD 14

- 21 eithe r once per day (s.i.d.) or a half dose twice per day (b.i.d.). Slides were quantified by at least 2 individuals blinded to the experimental

8 Mammary gland whole mount round robin summary. 2009; http://www.silentspring.org/pdf/our_research/ MammaryGlandWholeMountRoundRobinSummary.pdfPage 41 of 210 treatment. Numbers in parentheses above the columns indicate group size.

0 1 5 20 100 0 5 10 15 20 25 A

(10) (10) (10) (9)

(9)

ATR treatment (mg/kg)Branches / 9 mm2

0 1 5 20 100 0 5 10 15 20 25 D

(11) (6) (10) (10) (9)

ATR treatment (mg/kg)Branches / 9 mm2

0 1 5 20 100 0 10 20 30 40 50 B (8) (9) (7) (9)

(6)

ATR treatment (mg/kg)Number of TEBs

0 1 5 20 100 0 10 20 30 40 50 60 E

(11) (6) (9) (8)

(9)

ATR treatment (mg/kg)Number of TEBs

0 1 5 20 100 0 5 10 15 C (9) (9)
(8) (10) (9)

ATR treatment (mg/kg)Distance (mm)

0 1 5 20 100 0 5 10 15 F (11) (9) (10) (10) (6)

ATR treatment (mg/kg)Distance (mm) s.i.d. b.i.d.Page 42 of 210 Figure 2. Subj ective scoring of the control and high dose mammary gland whole mounts analyzed in Figure 1. Slides were scored by two individuals based on the system described by Enoch et al. (2007).

s.i.d. b.i.d. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 control 100 mg/kg

Dosing regimenSubjective score

A.2.16 Goldman J, Davis L, Murr A, Cooper R. (2011). The Influence of 1, 2, and 4 Days of Atrazine Treatments on the LH Surge in Ovariectomized/Estradiol

- Primed Rats: Preliminary Report to the Office of Chemical Safety and Pollution Prevention. Internal Report USEPA

The purpose of this study was to investigate the impact of 1, 2, or 4 days of atrazine exposure on the LH surge and circulating progesterone (one of the hormones involved in the regulation of the LH surge) levels. While a brief increase in progesterone levels on the day of proestr ous acts in conjunction with estradiol to trigger the LH surge, a prolonged period of elevated progesterone results in an attenuation of the LH surge. This is in fact, one of the basic principles used in the production of oral contraceptives.

Nine to elev en regularly cycling Sprague Dawley rats were bilaterally ovariectomized and implanted with an estradiol benzoate capsule (4 mg/ml in sesame oil). Three days after surgery, blood was collected at 1400, 1600, and 1800 hours from the lateral tail vein. At 2000 hours, the animals were sacrificed and trunk blood collected. Prior to blood collection, these animals had been exposed to 0 or 100 mg/kg/day atrazine (97% a.i. purity) by gavage once daily (at 1300 hours) for 1, 2 or 4 days. The group that received only 1 day of atrazine exposure was dosed at 1300 hours on the day of sacrifice; the group exposed for 2 days was dosed for the last two days before sacrifice, while the group exposed for 4 days received atrazine every day beginning the day before the ova riectomy. Measurements of circulating LH level were conducted using a radioimmunoassay with materials from the National Hormone and Peptide Program. Estradiol and progesterone levels were measured with a Siemens Healthcare Diagnostic antibody

- coated tube kit.Page 43 of 210 For animals receiving a single dose of atrazine on the day of sacrifice a statistically significant (p<0.01) increase in progesterone levels were observed one hour after dosing. However, progesterone levels returned to background ( i.e., comparable t o control) 3 hours after exposure. An LH surge is observed (manifested as an increase in both peak amplitude and area under the curve) in the animals receiving a single dose of atrazine. In contrast, 4 days of atrazine exposure did not change progesteron e levels from background. Without the physiological increase in progesterone, the LH surge was attenuated in these animals both in terms of peak amplitude and area under the

curve.
Reviewers Comments: This is a well conducted study that begins to invest igate the potential impact of single exposures to atrazine on the organism’s hormonal milieu. The data show that a single day of exposure elicits a marked increase in progesterone resulting in an LH surge similar to what is observed in intact cycling fema le rats. Given that the rats in this study had been ovariectomized prior to the atrazine exposure, the increase in progesterone seen is due to a rise of its synthesis in the adrenal glands. The observation that the elevated progesterone levels are observ ed one hour after a single exposure to atrazine and resolved by 3 hours suggests that atrazine’s effect on the adrenal glands is rapid and transient. The toxicological relevance and relationship between these changes in progesterone and LH and potential ad verse health outcomes is unclear. In addition,100 mg/kg/day is not an environmentally relevant dose thus a dose

- response for these findings would be informative in the future.

A.2.17 Coder, P. S. (2011) An Oral (Gavage and Dietary) Study of the Effects

o f Atrazine on the Spontaneous Luteinizing Hormone Surge in Female Sprague

- Dawley Rats, WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. Study Number: WIL

- 639080

In an oral gavage and dietary study the purpose was to evaluate the effects of atrazine exposure on the spontaneous luteinizing hormone (LH) surge in intact female Sprague Dawley rats following a distributed dose (diet) versus bolus (gavage) exposure over a 4

- day estrous cycle. In addition, the potential immun otoxic effects of atrazine were evaluated following distributed dose (diet) versus bolus (gavage) exposure after a minimum of 28 days of exposure.

NOTE* The previously submitted study by Coder, P. S. (2010) An Oral (Gavage) Immunotoxicity and Hormone Eva luation Study for the Effects of Atrazine on Male Rats concluded that treatment of the male rats with ATR orally for 28 days did not ultimately produce adverse biologically relevant effects on the AFC and NK assay. This current study assessed other compon ents of the immune system for their function and has concluded that no functional deficits in humoral and innate immunity occurred. These studies in combination have shown equivocal evidence of toxicity to the immune system and further the toxicology datab ase for atrazine is complete.Page 44 of 210 STUDY DESIGN:

For immunotoxicity assessments in Phase II, 2 positive control groups were also assigned to study. Animals in positive control group 1 (Group 8; Antibody

- Forming Cell [AFC] assay) were administered cyclophosphamide (50 mg/kg/day) once daily by intraperitoneal injection for 4 consecutive days prior to the day of scheduled euthana sia. Animals in positive control group 2 (Group 9; Natural Kill Cell [NKC] activity assay) were administered approximately 1.0 mL of diluted Anti Asialo GM1 by intravenous injection at approximately 24 hours prior to the scheduled euthanasia. The positive control groups were composed of 10 females each. Atrazine, was administered to female Crl:CD(SD) rats for comparison between distributed dose (dietary) versus bolus (oral gavage) exposure to atrazine. This study was divided into 2 phases (Phase I and Phase II ), and each phase consisted of 2 cohorts (Cohort A and Cohort B). Animals were administered the test substance over one 4

- day (or 5

- day) estrous cycle in Phase I for luteinizing hormone (LH) surge assessments, and selected animals from Phase I continued to be administered the test substance for a minimum of 28 days (up to 36 days of exposure) in Phase II for immunotoxicity assessments. Within each phase, animals in Cohort A were exposed to the test substance in the diet and animals in Cohort B were administ ered the test substance via oral gavage. In Phase I, female rats received the test substance on a continuous basis in the diet (Cohort A) or orally by gavage once daily (Cohort B) over one 4

- day (or 5

- day) estrous cycle. In Phase II, selected females fro m designated groups in Phase I continued to receive the test substance on a continuous basis in the diet (Cohort A) or orally by gavage (Cohort B) following completion of Phase I.

RESULTS: The results revealed no treatment related deaths. No test substance

related clinical findings were noted at any dose level during Phase I in Cohorts A or B. In Cohort A (dietary exposure), the av erage quantities of test substance consumed during Phase I (study days

- 1 to 3) in the 0, 30, 100, and 500 ppm groups were 0, 3, 9, and 40 mg/kg/day, respectively. Lower mean body weight gains were noted in the 500 ppm group during the first 3 days of expo sure, resulting in a lower mean body weight change for the overall Phase I treatment period (study days

- 1 to 4). In addition, mean body weights in the 500 ppm group were 4.1% to 5.1% lower than the control group during study days 1

- 4. Mean daily food con sumption in the 500 ppm group was lower than the control group throughout the Phase I treatment period, primarily as the result of decreased food consumption during the dark photoperiods. Mean body weights, body weight gains, and food consumption in the 30 and 100 ppm groups were unaffected by test substance exposure during Phase I.

In Cohort B (oral gavage administration), lower mean body weight gains were noted in the 50 mg/kg/day group during study days 0

- 2, resulting in a lower mean body weight change for the overall Phase I treatment period (study days 0 to 4). These lower mean body weight gains did not have a remarkable effect on mean body weights at 50 mg/kg/day. Mean food consumption in the 50 mg/kg/day group was slightly lower than the control grou p during study days 0

- 3 and for the overall Phase I treatment period (study days 0

- 4). Mean body weights, body weight gains, and food consumption in the 0.75, 1.5, 3, 6, and 10 mg/kg/day groups were unaffected by atrazine administration during Phase I. Bo lus (gavage) administration of atrazine for 1 complete estrous cyclePage 45 of 210 resulted in prolongation of the estrous cycle at 50 mg/kg/day. Five of the 21 females in the 50 mg/kg/day group exhibited a 5

- day estrous cycle during the Phase I treatment period. This prolongation of the estrous cycle from 4 to 5 days in the 50 mg/kg/day group was considered to be a result of atrazine administration. In addition, distributed dose (diet) exposure to atrazine at 30, 100, and 500 ppm had no effects on the length of the es trous cycle.

Atrazine, when administered via oral gavage at a dose of 50 mg/kg/day for 4 consecutive days was sufficient to inhibit the spontaneous LH surge in intact female rats, as indicated by decreased values for peak LH and area under the curve (AUC)

. An equivalent distributed dose of atrazine delivered in the diet (500 ppm) had no effect on the spontaneous LH surge, suggesting that inhibition of the LH surge is a consequence of achieving peak levels of atrazine above a currently undefined threshold. During Phase II, in Cohort A, the average quantities of test substance consumed in the 0, 30, and 500 ppm groups were 0, 3, and 51 mg/kg/day, respectively, for the AFC animals (study days 5

- 28) and the NKC animals (study days 5

- 30). No test substance

rel ated clinical findings were noted at any dose level during Phase II in Cohort A. Lower mean body weight gains/losses were noted intermittently throughout the treatment period which resulted in a statistically significantly lower mean body weight gain

- not ed in the 500 ppm group during the overall Phase II treatment period (study days 5

- 28 for AFC animals and study days 5

- 30 for NKC animals) evaluation. The lower mean body weights noted at 500 ppm during Phase I generally continued during Phase II (up to 7. 4% and 10.2% lower for the AFC and NKC assay animals, respectively). The reductions in mean food consumption noted in the 500 ppm group during Phase I were not sustained during Phase II. Mean body weights, body weight gains, and food consumption in the 30 ppm group were unaffected by test substance exposure during Phase II.

There were no test substance

- related, toxicologically significant effects on hematology parameters at any dose level or exposure route (dietary or oral gavage) in the NKC animals. Trea tment with atrazine via dietary or oral gavage exposure for a minimum of 28 days did not produce biologically relevant effects on the humoral or innate immune components of the immune response, when evaluated using the AFC assay and the NKC activity assay. There were no effects in absolute or relative spleen or thymus weights or spleen cell numbers (non

- Atrazine treated groups) from non

- immunized animals (NKC animals) treated with atrazine as compared to the respective (dietary or oral gavage) vehicle contr ol group.

Levels of urinary corticosterone were elevated in all groups during the second 6

- hour collection interval (9

- 15 hours) compared to the first 6

- hour collection interval (0

- 6 hours), but the elevation during the 9

- 15 hour interval was statistically significantly less in the 50 mg/kg/day group compared to the control group. There was no effect on the diurnal rise in urinary corticosterone in the 3 or 6 mg/kg/day groups. Atrazine did not alter circulating levels of prolactin, progesterone , corticosterone,Page 46 of 210 androstenedione, aldosterone, estrone or estradiol when administered via the diet (Cohort A) or by oral gavage (Cohort B) for 28 days.

CONCULSIONS: Lower body weight gains (or body weight losses) and/or reduced food consumption were obs erved following distributed dose (diet) exposure to atrazine at 500 ppm and bolus (gavage) exposure to atrazine at 50 mg/kg/day. In addition, administration of atrazine via bolus (gavage) exposure resulted in prolongation of the estrous cycle at 50 mg/kg/d ay, but there was no effect on estrous cycle length at a distributed dose (diet) of 500 ppm. Treatment with atrazine via dietary or via oral gavage exposure for a minimum of 28 days did not produce biologically relevant effects on the humoral or innate im mune components of the immune response, when evaluated using the AFC assay and the NKC activity assay. Based on the results of this study, the

no

- observed

- effect level (NOEL) for suppression of the immune system in female Crl:CD(SD) rats administered atra zine for 28 days via the diet or orally by gavage was 500 ppm and 50 mg/kg/day, respectively.

Bolus (gavage) exposure to atrazine for 4 consecutive days at a dose of 50 mg/kg/day was sufficient to impair the spontaneous LH surge in intact female rats. An equivalent distributed dose (dietary) was ineffective in blocking the surge of LH. When these animals were maintained on the same treatment paradigm for 28 days, atrazine did not alter circulating levels of prolactin, progesterone, corticosterone, androste nedione, aldosterone, estrone or estradiol, but there was a decrease in the diurinal rise (9

- 15 hours) of urinary corticosterone at a dose of 50 mg/kg/day. The NOELs in this study are based on the design using distributed (diet) or bolus (gavage) doses of atrazine up to 500 ppm (diet) or 50 mg/kg/day for 4 or 28 consecutive days.

Endpoint (ppm) (mg/kg/day) Body weight/food consumption 100 10 Immunotoxicity (humoral,innate) 500 50

Estrous cycle 500 10 LH surge attenuation 500 10 Plasma progesterone, prolactin, corticosterone, androstenedione, Aldosterone, estrone, estradiol NA 50

Urinary corticosterone NA 6

EPA Reviewer Comments: Results seem vastly similar to what was presented at previous SAPs and therefore conclusions to that of the registrant are also similar. Dietary dosing having no effect is not new knowledge. A statement regarding “re

bleeding" the animals if they were pro the first time they tried raises concerns. Since this issue could be problematic, further review is warranted to see how many rats were treated this way. The immunotoxicity component to this study provided little to know new knowledge on the lack of an effect on the humoral or innate components of the rats exposed.Page 47 of 210 A.2.18 Coder, P. S. (2011) An Study of the Effects o f Short

- Term Atrazine Exposure on the Estrogen

- Induced Luteinizing Hormone (LH) Surge in Ovariectomized Young

- Adult Sprague Dawley Female Rats, WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. Study Number: WIL

- 639091

In an oral gavage study the purpose was to assess the effects of short

- term atrazine exposure on the estrogen

- induced luteinizing hormone (LH) surge in ovariectomized young adult female Sprague

- Dawley rats following oral administration over 4, 8, or 14 days.

STUDY DESIGN: Atrazine was administered in 1% methylcellulose in deionized water by gavage to ovariectomized female Crl:CD(SD) rats. This study was divided into 3 cohorts (A, B, and C), and each cohort was composed of 4 groups (dose levels) of 25 ovariectomized females per dose. Dose levels were 0 (vehicle only), 6.5, 50, and 100 mg/kg/day administered at a volume of 5 mL/kg. The test substance or vehicle was administered for 4, 8, or 14 days to Cohorts A, B, and C, respectively, prior to collection of blood sam ples for LH evaluation; all animals received an additional dose the following day for possible assessment of atrazine plasma levels. The females were approximately 12 weeks of age at the beginning of treatment. Ovariectomies were performed on all females 3 days prior to the scheduled day of blood collection for LH assessment (study days 0, 4, and 10 for Cohorts A, B, and C, respectively). Blood samples for plasma atrazine analysis were collected from up to 12 females/group/time point at 30 minutes or appr oximately 4 hours after dose administration on the day following LH blood collection (study days 4, 8, and 14 for Cohorts A, B, and C, respectively).

RESULTS: The results revealed no treatment related deaths. Clinical findings related to treatment consi sted of red material around the nose and yellow material on the urogenital area in the 6.5, 50, and/or 100 mg/kg/day groups (Cohorts A and C), and salivation in the 100 mg/kg/day group of Cohort C. Mean body weight losses and lower mean body weights and f ood consumption were noted in the 50 and 100 mg/kg/day groups during the treatment period for all cohorts (study days 0

- 4, 0

- 8, and 0

- 14 for Cohorts A, B, and C, respectively). Females generally exhibited 4

- 5 day estrous cycles during pretest (all cohorts ). Vaginal lavages were discontinued beginning on the day of ovariectomy. In Cohort C, 4 and 5 females in the 50 and 100 mg/kg/day groups, respectively, exhibited 6

- 7 day estrous cycles, compared with no females in the control group. Estrous cycle length c ould not be evaluated in Cohort A (ovariectomy occurred on the first day of treatment) or in Cohort B (due to the limited number of days [4] prior to ovariectomy). There were no treatment

- related macroscopic findings at the scheduled euthanasia, and mean organ weights in all cohorts were generally similar across all groups, including the control group. Statistically significantly lower LH levels (peak concentration and AUC values) were noted in the 50 and 100 mg/kg/day groups with respect to the control g roup following 4 days of atrazine administration (Cohort A). Although LH levels in the 6.5 mg/kg/day group females in Cohort A were significantly lower at 13 hours post lights

- on, there was no effect on peak concentration or AUC and hence the effect noted at this single time point was not considered adverse. Following 8Page 48 of 210 and 14 days of atrazine administration, the 100 mg/kg/day group females (Cohorts B and C respectively) had significantly lower LH levels (peak concentration and AUC values) as compared to th e control group. Although mean LH levels in the 50 mg/kg/day group females in Cohort C were statistically significantly lower at 11 and 13 hours post lights

- on, there was no effect on peak LH level or AUC and hence the effect noted at 50 mg/kg/day followin g 8 and 14 days of exposure was not considered adverse

- Although the study indicates that a 1

- way ANOVA for peak LH measures revealed a significant effect of treatment (p<0.001), and a 1

- way ANOVA of LH AUC also revealed a significant effect of treatment (p=0.00311).

CONCULSIONS: Treatment

- related clinical findings were noted at all dose levels in Cohorts A and C but not in Cohort B. The incidence of these clinical observations in the 100 mg/kg/day groups of Cohorts and C were higher than the other treat ment groups. Treatment

- related decreases for body weight and food consumption parameters were noted for the 50 and 100 mg/kg/day groups during the treatment period for all cohorts. A treatment

- related effect in the number of animals with an increased cycl e length (6

- 7 days versus 4 days in pretest) was noted during the 10

- day treatment period prior to ovariectomy in the 50 and 100 mg/kg/day groups (Cohort C). Significantly lower LH levels (peak concentration and AUC values) were noted in the 50 and 100 mg /kg/day groups following 4 days of treatment (Cohort A), but only in the 100 mg/kg/day groups following 8 or 14 days of treatment (Cohorts B and C, respectively). The registrant determined no

- observed

- effect levels (NOELs) in this study are based on the d osing design in which 6.5, 50, and 100 mg/kg/day atrazine was administered for 4, 8, or 14 days to Cohorts A, B, and C, respectively (found in Table I).

Table I. Endpoint Cohort A (mg/kg/day) Cohort B (mg/kg/day) Cohort C (mg/kg/day) Clinical observations* 50 100** 50 Body weight/food consumption 6.5 6.5 6.5
Estrous cyclicity ND ND 6.5 LH surge* 6.5 50 50 Macroscopic exam 100 100 100

*No

- observed

- adverse

- effect level (NOAEL).

**No test substance

- related clinical findings noted in Cohort B.

ND

- Not determined due to insufficient number of days of assessment prior to ovariectomy.

EPA Reviewer Comments: The dosing formulations were visually inspected by the Study Director and therefore, stability and homogeneity analyses were not conducted. In Cohort A, one rat per dosing group (control, 6.5, 50, and 100 mg/kg/day) died on the day of surgery (study day 0), this seems questionable as well as the fact that of those animals that died, one from each the control and 50 mg/kg/day group were found to have both ovaries present. Section 3.4.3 Euthanasia of Unselected Females indicates that there were 24 females /group versus the before mentioned 25/group. In Cohort B,Page 49 of 210 one female in the 50 and 100 mg/kg/day groups were found dead about 4 hours after surgery while none were reported to have died in Cohort C. This is inconclusive since it doesn’t indicate whether the older the females were they either could withstand the surgery more so, or did the handlers get better with their technique over time? In Cohorts A and C clinical signs were found to be treatment related and were shown as increased incidences of red m aterial around the nose, (eyes, and mouth); yellow material on or around the urogenital area within 2 hours of dose administration. While in Cohort B these same clinical findings appeared but because they occurred in ‘single’ animals and in ‘similarity’ t o the control group were NOT considered dose related. Also found was hair loss, scabbing on forelimbs

– all effects considered not dose

- related since they mimicked what was seen in Cohort B. The analysis of LH levels showed that Cohorts A, B, and C with all doses there was a decrease in the estradiol induced LH surge in the afternoon of the 4 th , 8 th , and 14 th day of treatment, compared to the control group, while decreased peak LH and AUC were noted in the 50 and 100 mg/kg/day groups but not the 6.5 mg/kg /day group for Cohort A and only noted in group 100 mg/kg/day for Cohorts B and C.

Also noted in Cohort C was a treatment related increase in the cycle length (6

- 7 days versus 4 days in the pretest) during the 10

- day treatment prior to the ovariectomy in the two high doses tested. This effect was not further elaborated upon, but could have reasonable significance related to the lengthening of the estrous cycle in females exposed to atrazine repeatedly at those dose levels (50 and 100 mg/kg/day) for this p articular duration (>7 days). Ultimately it brings up the question as to whether in acute situations is the LH suppression truly adverse?

The NOAEL for LH surge suppression is found to be 6.5 mg/kg/day (chosen as a no affect level) for this study during 4 days of exposure. The LOAEL of 50 mg/kg/day was true for clinical observations at this duration.

A.2.19 Coder, P. (2011d) Atrazine: An Oral (Gavage and Dietary) Study of the Effects of Atrazine on Fertility and Reproductive Performance in Female Long Evans and Sprague

- Dawley Rats: Final Report. Report Number: WIL/639090, Task No. TK0052068. Unpublished study. Performing Laboratory: WIL Research Laboratories, Inc. 966 p. Completion Date: April 6, 2011. MRID 48470001.

Summary:

This study compared the effects of atrazine exposure on ovulation, fertility, and reproductive performance in Sprague

- Dawley (SD) and Long Evans (LE) intact female rats following diet versus gavage exposure over a 4

- day estrous cycle period.

Study Design: Twelve

- week old fem ale rats (25/dose level) with repeated 4

- day estrous cycles during the pretest period were exposed to atrazine (97.5%) for 4 days (or 5 days) via oral gavage up to 100 mg/kg/day (Cohort A: SD rats, doses 0, 12, 50, 100 mg/kg/day and Cohort B: LE rats, dose s 0, 1.5, 3, 6, 12, 50, 100 mg/kg/day) or via thePage 50 of 210 diet up to 1460 ppm (Cohort C: LE rats, doses 160, 660, 1460 ppm (0, 11, 35, 51 mg/kg/day). For Cohorts A and B, the vehicle and atrazine were administered orally via gavage once daily, beginning lights

- ON (0500 hours) on the first day of the estrous cycle (day of estrous; study day 0) and continuing over 1 complete 4

- or 5

- day estrous cycle. Females in Cohort C were given the control and test diets ad libitum beginning at lights

- OFF (1900 hours) on the 4 t h day of the previous estrous cycle (study day

- 1) and continuing over the next complete 4

- or 5

- day estrous cycle.

Vaginal lavages were performed daily during the pre

- treatment and treatment periods to determine the estrous cycle. Only females that exhi bited 4

- day estrous cycles repeatedly throughout the pretest period were assigned to the study. On the last day of treatment, females were paired with untreated males. Animals were paired for breeding prior to 1200 hours as the females were expected to be in proestrus that afternoon and in estrus the following morning. If evidence of mating was noted, the day was designated as day 0 of gestation. If evidence of mating was absent, the stage of estrous was recorded. If the female was in estrus, the female w as separated from the male without further opportunity for mating. If the female was in diestrus the morning following the initial pairing, the female was re

- paired with an untreated male rat that successfully mated. In the absence of mating on the second day, the stage of estrous was noted and the female was separated from the male without further opportunity for mating.

Clinical observations were recorded. Body weight and food consumption were recorded daily during treatment, on GDs 0, 3, 6, 9, 12, 15, 18 and 20 for females with evidence of mating. A laparohysterectomy was performed on GD 20 for each female with evidence of mating.

Functional endpoints (ovulation, fertility, and reproductive performance) were evaluated.

Results:

There were no survi val issues noted in the study for Cohorts A, B or C. No treatment

related clinical observations were noted for Cohorts A or B. Decreased defecation and small feces were noted in the 1460 ppm dose group (Cohort C) during the exposure period (study days

- 1 to 3). These findings were considered treatment

- related and correlated with a low mean food consumption observed during the treatment period.

During the treatment period, treatment related body weight losses and/or reduced body weight gains with correspon ding reductions in mean food consumption were noted at the two highest doses of atrazine administered (50 and 100 mg/kg/day in SD (Cohort A) and LE rats (Cohort B) and 660 and 1460 ppm in LE rats (Cohort C)). These effects did not affect mean maternal weig hts during gestation in any of the cohorts, which were comparable to controls by GD 20.

For Cohorts A, B and C, atrazine treatment did not affect the rat estrous cycle in either LE or SD rats.Page 51 of 210 Female reproductive performance (mating, fertility, or conc eption indices), the number of corporal lutea or implantation sites, intrauterine growth and survival or external fetal morphology in intact female SD or LE rats were unaffected by gavage doses up to 100 mg/kg/day in LE or SD rats or by dietary doses up to 1460 ppm (51 mg/kg/ay) in LE rats.

Reviewer’s comments: The underlying assumption in this experiment was that the animals would be dosed with an LH suppressing dose of atrazine. The animals were then bred to see if there were any affects on ovulation, fertility, and reproductive performance. This was done using two strains of rats via gavage and dietary exposures. The study authors concluded that “"Female reproductive performance (mating, fertility, or conception indices) was unaffected by bolus doses up to 100 mg/kg/day in LE or SD rats or by distributed doses up to 1460 ppm (51 mg/kg/day) in LE rats. Furthermore, mean numbers of corpora lutea, mean litter proportion of pre

- implantation loss, and the mean number of implantation sites were unaffected b y treatment. Intrauterine growth and survival and external fetal morphology were unaffected by atrazine administration. These results indicate that the magnitude of the atrazine

- induced preovulatory surge suppression shown in previous studies was not suffi cient to influence ovulation or reproductive performance of SD or LE female rats as evaluated in this study as the doses tested". There are some elements that confound the conclusions reached by the researchers. While it is not unreasonable to think that the atrazine induced LH suppression occurred at some of these doses, the LH plasma levels were not measured. A primary concern of this study was that the design did not really provide an opportunity to test the potential adverse effects of atrazine on th e endpoints examined. The assumption was that if atrazine decreased LH then this decrease would lead to some identifiable adverse outcome, at least in the high

- dose, gavaged animals. However, the design of this study did not allow for a true test of the assumption. For example, it is well

- known that if the spontaneous LH surge is compromised by pharmacological treatment (e.g., phenobarbitol, the usual positive control in such studies) and the female is mated later on the day of vaginal proestrus (as was done in the current study), mating itself will not be altered (because you have not interfered with the steroid hormones regulating sexual behavior) and that the act of mating (i.e., coital stimulation of the vaginal cervix) induces an LH surge (they have become reflex ovulators) and normal pregnancies will ensue (Tyler and Gorski 1980 Tyler, J and Gorski R. 1980. Temporal Limits for Copulation

- Induced Ovulation in the Pentobarbital

Blocked Proestrous Rat. Endocrinology. Vol. 106, No. 6: 1815

- 1819; Cooper et al. 1994 Cooper, R. Barrett, M, et al. 1994. Pregnancy Alterations Following Xenobiotic

- Induced Delays in Ovulation in the Female Rat. Fundamentals and Applied Toxicology. 22, 474

- 480.). Thus, it should be noted that even if the atrazine induced LH sup pression had occurred, the mating itself may have produced a surge and this alone may be responsible for the lack of any adverse effects on pregnancy in this study.

A.3 REPRODUCTIVE EF FECTS IN MALES

Summary: Reproductive/Developmental Toxicity, Delayed Puberty and ProstatitisPage 52 of 210 Since the IRED was finalized in 2003, several studies have been conducted assessing the effect of atrazine exposure on male reproductive function and development. Research has focused on a wide array of possible adverse reproduct ive effects including delayed puberty onset, prostatitis and changes in hormonal and andrology parameters.

There have been a number of studies demonstrating that atrazine delays male puberty in different strains of rats following both peripubertal and p erinatal exposure (Stoker et al. , 2000; Friedmann, 2002 ;Trentacoste et al., 2001; Rayner et al. , 2006 and Rosenberg et al., 2008).

The primary data collected following the male peripubertal exposures and examination of puberty outcomes (Stoker et al. , 2001; Friedmann et al. , 2002, Trentacoste et al.

, Pogrimic et al. , 2009) is consistent with the effect of atrazine on the central nervous system ( i.e. , suppression of GnRH/decreased pituitary secretion of LH) and subsequent alterations in the hormonal control of pubertal development (Cooper et al. , 2000). Decreased LH secretion would lead to insufficient stimulation of the gonads during puberty to prod uce the necessary steroids for androgen

- dependent maturation of the sex accessory tissues in the male. Therefore, the delayed growth of the reproductive tract in the male supports the reported alteration of the hypothalamic control of pubertal progression

. A reduction in testosterone levels following atrazine exposure has been reported in a number studies in mammals, as well as other species revealing a consistency in the effects of atrazine on androgens. It is well known that the development of the si ze of the penis and cornification of the epithelium of the prepuce and preputial separation in immature rats are regulated by androgens (Marshall, 1966). A decrease in testosterone secretion during the juvenile period can delay preputial separation (Lyons et al. , 1942) and reduce the size of the androgen

- dependent tissues, such as the ventral prostate and seminal vesicles. Normally, testosterone levels rise gradually from PND 20 to 40, and abruptly double by PND 50 (Matsumoto et al. , 1986; Monosson et al. , 1999).

In the male rat, atrazine exposure resulted in delays in the onset of puberty, as determined by assessment of preputial separation (PPS) (Table 4). Stoker et al. (2001) found that peripubertal exposure to atrazine delayed puberty for 1.5 to 2 .5 days following exposure to 12.5

- 200 mg/kg atrazine. The NOAEL for the effect on puberty in the male Wistar rat was 6.25 mg/kg/day and the LOAEL was 12.5 mg/kg/day. They also observed significant decreases in the growth of the reproductive tract, nam ely the seminal vesicles and prostates, of the affected male rats at slightly higher doses than the doses required to delay puberty (NOAEL = 25 mg/kg). At PND 45, intratesticular testosterone was decreased by 200 mg/kg. In the studies examining the Sp rague Dawley (SD) rat (Trentacoste et al. , 2001), pubertal onset was delayed by 3 days and 4 days following exposure to 100 and 200 mg/kg atrazine, respectively, with decreases in ventral prostate and seminal vesicles at the same doses (NOAEL = 50 mg/kg). Friedmann (2002) found a decrease in intratesticular and serum testosterone at 50 mg/kg/day and 200 mg/kg/day following exposure to the peripubertal male SD rat.Page 53 of 210 Thus, all three studies found decreases in either serum or intratesticular testosterone

a nd Trentacoste et al. also found a decrease in LH at the 200 mg/kg/day dose.

Ventral prostate, seminal vesicles, lateral prostates and epididymal weights are significantly reduced at doses just above the doses which delay preputial separation (Stoker et al. , 2001). These effects provide further evidence of a decrease in androgen stimulation. Decreases in androgen hormone concentrations also correspond with the effects observed on pubertal endpoints. For example, Stoker et al. , 2001 demonstrated that in tratesticular testosterone was significantly decreased following atrazine exposure peripubertally. This has corresponded to the work of others showing that serum testosterone is decreased by doses of 50, 100 and 200 mg/kg in a different strain of rat (SD) when dosed during a similar period of PND 22 to 47 (Trentacoste et al. , 2001; Friedmann, 2002) as shown in Table 4.

Table A

- 2 : Strain and Dose Comparison in Male Rats dosed peripubertally with atrazine

EXPOSURE STRAIN EFFECT N OAEL/LOAEL REFERENCE PND 23 to 53 Wistar Delayed PPS 6.25/12.5 Stoker et al. , 2001 PND 22 to 47 SD Delayed PPS 50 lowest dose/100 Trentacoste et al.

, 2001 PND 23 to 53 Wistar Decreased VP and SV 25/50 Stoker et al. , 2001

PND 22 to 47 SD Decreased VP and SV 50 lowest dose/100 Trentacoste et al.

, 2001 PND 23 to 53 SD Decreased intra

testicular T on PND 45 150/200 Stoker et al. , 2001

PND 22 to 47 SD Decreased T on PND 47 ND/50 Trentacoste et al.

, 2001 PND 22 to 47 SD Decreased test. and serum T on PND 47 ND/50 Friedmann, 2002

PND 23 to 50 Wistar Decreased T/DHT on PND 50 50/200 Pogrimic et al.

, 2009

The study by Stoker et al. , 2002 also examined the effect of the atrazine metabolites, DACT, DIA and DEA on pubertal development in the rat. In this study, equimolar doses of the metabolite were compared to the effect seen with atrazine in the earlier study (Stoker et al. , 2000). PPS was significantly delayed by DEA and DIA at doses ≥ 25 mg/kg/day and by DACT at doses ≥12.5 mg/kg/day. When the males were sacrificed on PND 53, treatments with DEA at atrazine equivalent dose (AED) ≥100 mg/kg/day, DIA at an AED ≥ 50 mg/kg/day, and D ACT at an AED of 200 mg/kg/day caused a significant reduction in ventral prostate weight, while only the highest doses of DIA and DEA resulted in a significant decrease in lateral prostate weight. Seminal vesicle weight was reduced by DEA at AEDs ≥25 mg/kg /day, and by DIA and DACT at AEDs ≥100 mg/kg/day AED. Epididymal weights were reduced in the DEA, DIA, and DACT at atrazine equivalent doses of 200, 200, and ≥100 mg/kg/day, respectively. Serum testosterone was reduced only in the males receiving the 2 h ighest doses of DIA. ThisPage 54 of 210 study confirmed that the chlorinated metabolites of atrazine also result in similar effects on pubertal development.

It should be mentioned that other factors, in addition to those changes within the HPG, can affect the timing of puberty in the male rat including nutritional status (leptin/body weight) and environmental (pineal gland/secretion of indole amines) influences. Nutritional status and body weight are known to have effects on puberty, which are suggested to reflect t he metabolic signals in the brain that serve as indices of the metabolic state. These metabolic alterations associated with weight loss or decrease in growth rate are inhibitory to the reproductive system and may be related to substances in the body that can alter the release of GnRH such as insulin, amino acids necessary for precursors of neurotransmitter synthesis and essential fatty acids (Ojeda and Urbanski, 1994).

The peripubertal exposure male study (Stoker et al. , 2001) showed decrease body weig ht at the 200 mg/kg dose, but a pair

- fed group was included to discern the effects of changes in growth. There were changes in puberty at doses in which no body weight decreases were observed ( ≥50 100 mg/kg/day in females and ≥12.5 mg/kg/day males). Howe ver, in the study with SD rats Trentacoste et al. , found that the food restriction group (receiving approximately 10% less food which was equivalent to the 100 mg/kg atrazine treatment food intake) showed the same effects on LH, testosterone and reproducti ve tract development. A recent paper examined the effects of food restriction on the endpoints in the pubertal male exposure period and found that reductions in body weight up to 10% did not influence pubertal onset or the growth of androgen dependent tis sues in the rat (Laws et al. , 2007).

Perinatally a single 100 mg/kg atrazine dose to the dam resulted in an effect on pup growth that may involve changes in milk quality, as the offspring showed a decreased weight of 7% on PND 4 (Rayner et al. , 2006). Work by Rosenberg and co

- workers (2008) showed that prenatal (GD14

- parturition) exposure to atrazine at doses ≥10 mg/kg/day led to a significant delay in preputial separation (1

- 3 days) while anogenital distance on PND21 was reduced at 75 and 100 mg/kg/da y. Measuring testosterone levels in both the serum and intratesticular fluid of PND 60 rats revealed that serum testosterone levels were decreased at doses ≥ 75 mg/kg/day while intratesticular fluid testosterone was reduced at doses ≥ 50 mg/kg/day. It sh ould be noted that all of the effects on PPS and AGD occurred in conjunction with nonsignifcant decreases in pup body weights (Rosenberg et al., 2008). These alterations of early postnatal body weight could play a role in the delay in puberty in these mal e offspring.

In summary, the MOA of altered pubertal progression in the rat and decreased androgen production by the testes is plausible in humans, as GnRH and the regulation of pituitary hormones stimulate the gonadal secretion of steroids and is critical for the init iation of the set of events and time course of the HPG regulation of puberty in the human. Thus, it is essential to take into consideration that all the effects on sexual maturation and altered androgen status reported after ≈ 30 days of exposure occur atPage 55 of 210 dose levels substantially higher ( ≥ 12.5 mg/kg/day) than those leading to the LH surge disruption which serves as the basis of the Agency’s current risk assessment.

Prostate effects including prostatitis are oftentimes attributed to atrazine exposure. T he increased incidence of prostatitis after atrazine exposure has been the subject of study for at least the last decade. Since 2003, there have been two in vivo studies to further explore this potential adverse health outcome.

First reported in 1999, an increase in prostatitis has been linked to the suppression of suckling

- induced prolactin release. After lactating dams are exposed to atrazine on PND1

- 4 at doses ≥ 12.5 mg/kg/day, the incidence of prostatitis in their offspring increased. At doses ≥ 2 5 mg/kg/day both the incidence and severity of the findings were increased. Notably, at 12.5 and 25 mg/kg/day the suckling induced release of prolactin was decreased in some dams while the 50 mg/kg/day dose level elicited a prolactin decrease in all femal es. Prolactin plays a crucial role in neonatal brain development particularly in the tuberoinfundibular dopaminergic system (TIDA). In the adult male, these neurons are responsible for the down

- regulation of prolactin release from the pituitary. As a con sequence of the decreased levels of maternal prolactin during the early lactational period, the offspring of these dams do not receive the perinatal amounts of prolactin necessary to activate the TIDA neurons. Therefore, TIDA’s regulation of prolactin in the adult male is impaired resulting in elevated prolactin levels which in turn lead to hyperprolactinemia. It is this elevated level of circulating prolactin in the adult males that has been linked to an increased incidence of prostatitis. Thus, an incre ased incidence of prostatitis in the offspring of dams exposed to atrazine during the critical time for TIDA neurons activation (first postnatal week) may attributed to decreases in circulating prolactin levels in the dam.

Using a cross

- fostering study de sign, Rayner et al. (2007) investigated if gestational exposure to atrazine could also lead to prostatitis in animals exposed in utero. Dams were exposed to 100 mg/kg/day atrazine from GD 15

- 19 and after birth the pups were

cross

- fostered resulting in the following grouping:

**•** ATR

- C: pups exposed to atrazine in utero only (born to atrazine

- treated dams but nursed by control females)

**•** C

- ATR: pups exposed to atrazine lactationally only ( i.e., born to control dams but nursing from atrazine

- treated dams)

**•** ATR

- ATR : pups exposed to atrazine both in utero and through the milk

**•** C

- C: pups born to and nursing from control dams

Consistent with the findings reported by Stoker et al. , both of the groups exposed postnatally ( i.e., C

- ATR and ATR

- ATR groups) exhibited indica tions of lateral prostate inflammation on (PND 120). No prostate inflammation was observed in the ATR

- C or C

- C groups suggesting that the early postnatal period encompasses the critical window of susceptibility to atrazine

- induced prostatitis. It should be noted, however, that these effects occur at doses higher than those eliciting disruption of the LH surge.Page 56 of 2 10 Based on the observation that atrazine exposure led to a decrease in testosterone production in rats, it has been suggested that this decrease wou ld in turn ameliorate prostate carcinoma development. Kandori and coworkers (2005), however, hypothesized that rather than being a direct hormone

- mediated effect, the decrease in prostate carcinogenesis is the secondary effect of a reduction in caloric in take. To test this hypothesis these researchers used a transgenic adenocarcinoma rat model typically used to study prostate carcinogenesis. Rats received either 33 or 61.6 mg/kg/day of atrazine for a period of 13 weeks while another group was placed on a calorie

- restricted diet. Atrazine treatment at the highest dose level (61 mg/kg/day) and dietary restriction elicited a slight suppression of prostate carcinogenesis. Unlike other researchers, testosterone levels, however, were not reduced by atrazine t reatment in the study conducted by Kandori et al. (2005). The authors theorized that, since testosterone levels were unaffected by atrazine treatment, the decrease in prostate carcinogenesis observed after atrazine exposure was due to reduced caloric inta ke (resulting from atrazine exposure) rather than hormonal disruption. It is important to know, however, that it has been demonstrated by other researchers that exposure to atrazine at the doses used in this study leads to decreases in testosterone. It is not clear if the discrepancy between this study and other studies that have demonstrated atrazine

- induced alterations of testosterone levels is due to differences in administration of atrazine (dietary vs. oral gavage). In addition, testosterone levels w ere measured only at the end of the study and were highly variable. Also, there was a potential problem with the homogeneity of the diets (concentrations of atrazine were 60

– 91% of target values). While this study suggests the possibility that prostate carcinogenesis may be a secondary effect to a reduction in caloric intake, a test system in which testosterone levels are altered would provide more definitive confirmation.

In summary, the data indicate that atrazine induces prostatitis at doses ≥ 12.5 mg/kg/day and that early postnatal exposure is a critical window of susceptibility to this effect

A.3.1 Abarikwu S., Adeslyan A., Oyeloja, T., Farombi, E. 2010. Changes in Sperm Characteristics and Induction of Oxidative Stress in the Testis and Epidi dymis of Experimental Rats by a Herbicide, Atrazine. Arch. Environ Contam Toxicol 58:874

- 882. DOI 10.1007/s00244

- 009

- 9371

- 2.

In this study, the authors investigated the effects of atrazine on oxidative stress in rat reproductive organs.

Adult male Wista r rats (10/group) were exposed to 0, 120, or 200 mg/kg/day atrazine (commercial product cotrazine 80WP, an 80% wettable powder) orally (dissolved in soya oil) for 7 or 16 days. Body weight, food intake, and reproductive organ weights were recorded. The fo llowing biochemical assays to measure oxidative stress were conducted on the testes and epididymides: catalase (CAT) activity, H 2 O 2 - mediated oxidation of Fe 2+ to Fe 3+ , superoxide dismutase (SOD), γ

- glutamyl transferase ( **γ** - GT), glutathione

- S

- transferase (GS T) activity, glutathione (GSH), lipid peroxidation (LPO), which was quantified as malondialdehyde MDA), ascorbate (AA) and protein content.Page 57 of 210 Testicular and epididymal sperm number and progressive sperm motility were measured. Morphological and viability of spermatozoa were examined. Daily sperm production (DSP) and DSP/gram testis were calculated.

At 200 mg/kg/day body weight was significantly decreased (p<0.05) compared to controls at 7 days ( ↓21%) and 16 days (↓19%). No decrease in body weight was seen at 120 mg/kg/day at either 7 or 16 days. Food intake was significantly decreased at 200 mg/kg/day at both 7 and 16 days.

Animals exposed to the high dose of atrazine (200 mg/kg/day) showed statistically significant (p<0.05) reductions in reproductive organ weights, including epididymis ( ↓25% absolute/↓12% relative at 7days; ↓29% absolute/↓17% relative at 16 days), seminal vesicle ( ↓41% absolute at 7 days; ↓28% absolute at 16 days) and prostate ( ↓38% absolute/↓48% relative at 7 days; ↓50% absolute/↓40% relative at 16 days). No changes in these organ weights compared to controls were observed at the low dose of 120 mg/kg/day. The absolute testicular weight remained unaffected at both dose levels, but the relative testicular weights were significantly de creased by 23

- 29% at both 7 and 16 days at both dose levels, however, this was not considered to be adverse since there was no change in absolute testicular weight..

Sperm motility, testicular sperm number, epididymal sperm number and daily sperm producti on were significantly (p<0.05) decreased at both dose levels and both time points. Significant increases (p<0.05) were seen in dead sperms and abnormal sperm rate at both dose levels and both time points.

There was a significant dose related increase in testicular GST and GSH activities during the 7 day study while SOD and CAT activities were similar to controls. The GSH activity was considered to be an adaptive response to oxidative stress. When atrazine treatment was increased to 16 days, CAT activity decreased in both dose groups in epididymis but remained unchanged in the testes, while SOD activity decreased in both dose groups in both the testes and epididymis. **γ** - GT was significantly increased in the testes at both doses and timepoints, while AA re mained unchanged. Epididymal LPO was significantly increased at both dose levels after 16 days. MDA levels in the testes were significantly increased at both dose levels at 16 days but not at 7 days. Hydrogen peroxide generation in the testes remained unc hanged in both dose levels after exposure for 7 or 16 days. According to the study author, the seminiferous tubules were normal except for some tubules with mild degeneration. No morphological changes were seen in the epididymis (data not shown),

Signific ant positive correlations were seen with the following: 1) testis and epididymal weights and testicular sperm number (TSN) and epididymal sperm number (ESN), respectively; 2) testicular and epididymal MDA levels and abnormal sperm rate; 3) MDA levels with testis SOD activity. Significant negative correlations were seen with the following: 1) testicular and epididymal MDA levels and sperm numbers and 2) epididymal MDA levels with CAT and SOD activities.Page 58 of 210 Comments: The results suggest that the observed bioch emical and spermatogenic aberrations may be due to oxidative stress in the rat testis and epididymis, however, the influence of secondary factors such as decreased body weight and food intake cannot be ruled out. There were only mild changes in the semini ferous tubules. The measures of oxidative stress were for the most part modest with emphasis on the high dose effects for a few measures. The overall functional significance of these changes remains unclear given the degree of change in the responses.

A. 3.2 Betancourt, M., Reséndiz, A., Casas, E. and Fierro, R. (2006) Effect of two insecticides and two herbicides on the porcine sperm motility patterns using computer

- assisted semen analysis (CASA) **in vitro**

. Repro. Tox. 22:508

- 512.

In this study the autho rs investigated the effects of four pesticides

– malathion, diazinon, fenoxaprop ethyl, and atrazine

– sperm viability, morphology, and motility pattern in porcine semen. An in vitro viability staining method was used to assess viability while a computer

- assisted

- analyzer (CASA) was used to study sperm mobility. 9

Semen samples were collected from 3 Duroc boars of proven fertility and the sperm checked for anomalies in motility or mo rphology. Only samples with sperm motility ≥ 85% and sperm abnormalities < 15% were used in the experiment. Sperm at a concentration of 25 x 10 6 sperm/ml, were placed in 4 well plate samples at a final concentration of 0, 50, 100, or 500 μM in a volume of 500 μl tris

- buffered medium [approximately 0, 5.4, 11, or 54 μg] and incubated for 1 hour at 37°C. For the viability analysis, 10 μl of the semen sample were placed on a slide and stained with 10 μl eosin

- nigrosin staining. Sperm that were viable did not incorporate the stain while dead spem were stained purple. For the motility evaluations, 8 μl of sperm sample were placed on a slide and observed using a CASA analyzer. Parameters measured by this device include mean velocity, linear velocity, curvi linear velocity, mean lateral head displacement, and frequency of head displacement.

Exposure to 11 or 54 μg of atrazine resulted in an 8% and 21% statistically significant (p<0.05) decrease in sperm viability, respectively. With respect to sperm motilit y, atrazine exposure at 11 or 54 μg led to a statistically significant decrease in progressive motility of 23 and 75%, respectively. Atrazine exposure appeared to affect the mean velocity and linear velocity of sperm. These parameters are indicators of s perm progression. In contrast, other parameters examined by the CASA that were not affected by atrazine ( e.g., mean lateral head displacement and frequency of head displacement) are indicators of sperm vigor.

The authors of this study conclude that atraz ine has an effect on sperm motility and, therefore, affect fertility. It should be noted, however, that no data are available from this study to corroborate this hypothesis. Interestingly, other researchers have reported that the outcomes of in vitro fer tilization (IVF) using sperm treated with up to 10 μM

9 In this evaluation only the results for atrazine will be discussedPage 59 of 210 atrazine were not affected. The authors also speculate that the effects in sperm motility may be a sequela of mitochondrial function disruption

– cellular respiration, in particular.

Reviewer’s Comme nts: Overall, the data suggests an effect of atrazine on sperm motility but not morphology or viability. The study, however, entails direct exposure of germ cells to atrazine without taking into account physiological conditions that may preclude the pesti cide from eliciting this effect in vivo . Given that atrazine is rapidly metabolized, it is likely that the metabolites and not the parent compound are the moieties reaching the sperm and testis. This consideration, however, was not accounted for by the s tudy authors. Moreover, no attempt was made by the researchers to investigate the consequences of these changes in sperm motility on reproductive health ( e.g., decrease in fertility). In the absence of these considerations, the data presented have little relevance to human health risk assessment

A.3.3 Friedmann, AS. 2002. Atrazine inhibition of testosterone production in rat males following peripubertal exposure. Reproductive Toxicology 16: 275

- 279.

Juvenile Sprague

- Dawley male rats (n=4

- 6/dose group) were administered 0 or 50 mg/kg/day atrazine (purity not stated) via gavage for either a 3

- day exposure (PND 46

- 48) or a 27

- day exposure (PND 22

- 48). For both exposure scenarios, animals were killed on PND 49 between 9 and 11 in the morning by decapitation . Testosterone (T) levels in trunk blood, interstitial fluid of testes and cell culture media were measured. [Note: The 50 mg/kg/day dose was based on a pilot study in which rats were dosed with 50 and 200 mg/kg for PND 22

- 48 and killed on PND 49. Both do ses lowered body weight, reduced serum T and interstitial fluid T.] For the in vitro treatment looking at testosterone production, Leydig cells isolated from rats on PND 49 were co

- cultured with 50 μg/ml or 232 μM atrazine in the presence of a maximally s timulating concentration of luteinizing hormone.

In the 27

- day in vivo exposure to 50 mg/kg/day, mean testosterone concentrations were significantly reduced by 38% in serum and 53% in intratesticular testosterone. Decreased body weight (7%) was seen in the 50 mg/kg/day group (215 g) compared to controls (230 g). In the 3

- day exposure to 50 mg/kg/day atrazine reduced both serum (57%) and intratesticular (58%) levels of testosterone. There was no significant decrease in body weight of the atrazine group compared to control in the 3

- day exposure treatment.

In vitro treatmen t of Leydig cells for 3 hours with atrazine culture medium resulted in significantly reduced testosterone production (35%) compared to controls. Atrazine also reduced testosterone levels in Leydig cells stimulated with db

- cAMP by 34%. However, atrazine di d not reduce testosterone production when cells were stimulated with hydroxycholesterol.

The author concluded that atrazine inhibits steroidogenesis in vitro and in vivo . The mode of action is via inhibition at the testicular level.Page 60 of 210 Friedmann argued agai nst the earlier paper by Trentacoste (he was a co

- author) that concluded the decrease in T was only the result of underfeeding. He did this by citing the drastic reductions in food intake needed to lower serum T. This argument was supported in the Stoker et al., paper mentioned above in which she measured serum T and interstitial T and found no difference in the males underfed to the 200 mg/kg group. Finally, the fact that the short

- term treated rats also had lower T values and these animals had not diff erent BWs also argues against the suggestion that all the hormone changes observed in these studies is a function of decreased weight.

In discussing his in vitro study Friedmann concluded that T production by the Leydig cell was decreased when exposed to atrazine. The mechanism for this, if repeatable, is unclear. Friedman did suggest that as the synthesis of testosterone in the presence of hydroxycholesterol was not impaired, the herbicide interferes with cAMP’s role in stimulating Leydig cells Furt hermore, it should be noted that unpublished data from the EDSP validation studies conducted at NHEERL, used much lower doses of atrazine in culture and measured T production in H295R cells and compared the results with similar treatment to primary Leydig cells. In both cases, T production was increased (see Higley et al., paper for data on H295R cells using doses ranging from 1

- 100 μM).

Reviewer’s Comments: In summary, a limited number or no measures of key androgen dependent tissues were examined in t he in vivo study presented in this paper, especially in the long

- term dosed rats. The in vitro data needs repeating with a range of doses.

A.3.4 Hase Y, Tatsuno M, Nishi T, Kataoka K, Kabe Y, Yamaguchi Y, Ozawa N, Natori M, Handa H and Watanabe H. (2008) Atrazine binds to F1F0

- ATP synthase and inhibits microchondrial function in sperm. Biochem Biophy Research Comm 366: 66

- 72.

This paper demonstrates the ability of ATR to alter human sperm motility in vitro , and tests hypotheses on the mechanism through which this might occur, specifically by decreasing ATP availability for sperm flagellar motion. In a series of biochemical tests, the authors present evidence that ATR can bind to F 1 F 0 - ATP synthase and thereby inhibit mitochondrial function (decreasing A TP available for sperm motion).

Note: The title is misleading because the authors did not actually determine effects on sperm mitochondria

– rather they used liver mitochondria. Sperm energetics are quite different from somatic cell energetics and spe rm mitochondria differ from somatic mitochondria in specific ways that should be discussed.

Approach: Ejaculated human semen samples were washed to recover motile sperm and the sperm were then cultured with atrazine (ATZ) and ATZ

- NH2, a modified version of ARZ needed for subsequent biochemical studies). Oligomycin, a sperm metabolic poison which uncouples ATP production in mitrochondria, was used as a positive control. Oligomycin inhibited % progressive sperm at 0.05 mM; ATZ inhibited at 0.3Page 61 of 210 and 1.0 m M but not 0.1 ( i.e. was less effective than the positive control). Sperm ATP levels were also measured and shown to be significantly decreased at the same concentrations of test compounds that inhibited % progressive motility. Motility assessments were made using established computer aided sperm analysis (CASA) methods.

Data rule out several early steps in ATP synthesis and make a solid case for ATR ability to bind the ATPase.

The authors conclude that this likely explains earlier reports that ATR decreases porcine sperm motility in vitro (as discussed in the preceding two papers). Importantly, they also conclude that “This study was motivated by the idea that ATZ applied to fields may cause a reduction of sperm count and motility to humans and oth er animals. However, the effect of ATZ on sperm motility was seen at >100 μM, exposure levels that probably do not result from normal use of the herbicide. It is, therefore, uncertain whether the observed phenomena are ecologically relevant.”

Reviewer ’s Comments: The relevance of these findings to risk assessment is questionable given that the concentration levels required to affect sperm motility were high and exceed likely exposure levels resulting from atrazine use in the environment. Moreover, the relevance of these ex vivo findings to the in vivo physiologically relevant conditions is also uncertain.

A.3.5 Kandori, H, Suzuki S, Asamoto M, et al. 2005. Influence of atrazine administration and reduction of calorie intake on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats. Cancer Sci: 96: 221

– 226.

The effects of a 13

- week dietary administration of 500 and 1000 ppm atrazine on the development of androgen

- dependent prostate cancer were investigated using male pr obasin/SV40 T antigen transgenic (TG) rats (Sprague

- Dawley genetic background, 6 weeks of age). Based on food consumption data, the corresponding doses in mg/kg/day were 33.0 mg/kg/day for the 500 ppm group and 61.6 mg/kg/day for the 1000 ppm group. Eac h treatment group contained 10 animals. A control group was included.

The rationale for using TG rats was based on previous findings that atrazine inhibited testosterone production in the Leydig cells directly of male rats. This would allow for an

exa mination of atrazine treatment on the development of androgen dependent prostate carcinomas. The authors noted that the TG rats have been established in their laboratory, and have developed well

- differentiated adenocarcinomas in the prostate at 100% inci dence before 15 weeks of age. Since the tumors are considered to be completely androgen dependent, the hypothesis was that exposure of atrazine to the TG rats would cause inhibition of testosterone production, resulting in reduced carcinoma development. Since atrazine has been reported to decrease body weights, a dietary

- restricted TG rat group was also included to assess the influence of reduction of calorie intake on the development of prostate cancer.Page 62 of 210 The authors reported that mean food consumption in the dietary

- restricted group was about 70% of that in the control group. At the end of 13 week treatment period, the following organs were subjected to histological examination: testes and accessory sex organs, ventral prostate, the dorsolateral prost ate including the urethra, and the anterior prostate/seminal vesicle complex. Serum testosterone levels were measured for every animal in each group using radioimmunoassay. Serum levels of atrazine and atrazine metabolites were measured for 3 animals pe r group using gas chromatography

- mass spectrometry. Immunohistochemistry for SV40 T antigens and androgen receptors were performed using avidin

- biotin

- peroxidase complex method. The ratios of prostate epithelial areas were evaluated morphometrically.

The authors reported that no deaths were observed. Body weights in the atrazine treated groups were significantly lower than in the control group. Bodyweights of the dietary

- restricted and 1000 ppm atrazine groups were similar, and significantly lower than control values throughout the treatment period. Food consumption was lower in the atrazine treatment groups compared with control group. In the dietary

- restricted group, mean relative ventral prostate weight was significantly decreased (25%) relati ve to controls. No other treatment

- related changes in relative organ weights in any treatment group were reported. Serum testosterone levels were not significantly different between the atrazine and dietary restricted groups compared with control group. Atrazine and its metabolites diaminochlorotriazine, desethylatrazine, and desisopropylatrazine were detected in the serum of the atrazine treated animals, but not in the control or dietary restricted groups.

Histopathological examination of the ventral , lateral, and anterior lobes of the prostate revealed lesions in all groups including controls. The authors described the lesions as marked epithelial proliferation with formation of irregular glands and luminal bridging to give a cribriform pattern in f ocal populations. The nuclei demonstrated enlargement and severe atypia. According to the authors, the lesions appeared compatible with well

differentiated adenocarcinomas in human cases and were therefore diagnosed as adenocarcinomas. These were observ ed in the ventral, lateral, and anterior lobes of the prostate in almost all rats. In the dorsal lobe, adenocarcinomas were observed in two of three rats of each group. The incidences of adenocarcinomas in any treatment group were not different from thos e of the control group. Foci within glands with less proliferation were also observed. These exhibited crowding of stratified epithelial cells with irregular spacing and occasional luminal bridging. Nuclear atypia were described as severe, however, basi c structures of the prostate glands were mostly maintained, comparable with prostatic intraepithelial neoplasias (PIN) of human cases.

Because almost the entire lobes of the prostate were occupied with tumor lesions at week 13, morphometric assessment r atios of the prostate epithelial area to the whole prostate tissue area were evaluated. Morphometric examination of the prostate epithelium revealed significant decreases in the ratios of the epithelial areas to whole prostate tissue areas in the ventral, dorsal, and lateral lobes in the dietary

- restricted group, and in the lateral lobe of the 1000 ppm atrazine group. The values in the dorsalPage 63 of 210 lobe in the atrazine groups showed a tendency to decrease (not statistically significant) in a dose

- dependent mann er. No significant effect was observed in the anterior lobe in any treatment groups.

SV40 Tag and androgen receptor expression were detected immunohistochemically in almost all cells of the prostate adenocarcinomas and PIN. No treatment

- related changes in the testes, epididymides, and seminal vesicles were observed. The authors concluded that dietary administration of atrazine for 13 weeks showed slight suppressive effects on the development of prostate cancer in the probasin

- SV40 T antigen transgenic rats, even though atrazine treatment did not affect serum testosterone levels. Based on the observation that there was equal or greater suppression of prostate neoplastic lesion development in the dietary

- restricted group, the authors also concluded that the observed atrazine

- related suppression of prostate cancer was probably caused by the decrease in calorie intake, rather than endocrine effects.

Revewer’s Comments: While this study suggests the possibility that prostate carcinogenesis may be a second ary effect to a reduction in caloric intake, a test system in which testosterone levels are altered would provide a more definitive confirmation.

A.3.6 Laws, S.C., Hotchkiss, M., Ferrell, J., Jayaraman, S., Mills, L., Modic, W., Tinfo, N., Fraites, M., St oker, T., and Cooper, R. (2009). Chlorotriazine Herbicides and Metabolites Activate an ACTH

dependent Release of Corticosterone in Male Wistar Rats. Tox. Sci. 112(1):78

- 87.

This study is a follow

- up to a previous set of experiments study in which these

r esearchers demonstrated that atrazine exposure results in an increase in steroidogenesis leading to increases in corticosterone, progesterone, and estrogens. In this study the investigators sought to characterize the temporal changes in adrenocorticostero id (ACTH), progesterone, and corticosterone in males receiving a single dose of atrazine, simazine, propazine or the chlorinated metabolites of these triazines (DIA, DEA,and DACT). 10 Male Wistar rats ( ≈ 50

- 60 days old) received 0, 5, 50, 100, or 200 mg/kg atrazine while animals exposed to DIA received 4, 10, 40, 80, or 160 mg/kg and DEA

- exposed animals received 173 mg/kg. DACT

- exposed animals received 3.37, 33.7, 67.5, or 135 mg/kg. The authors state that the maximum dose for each metabolite was the mola r equivalent of atrazine at 200 mg/kg. Animals were sacrificed precisely 5, 15, 30, 60 or 180 minutes after exposure. ACTH, corticosterone, progesterone, and prolactin plasma levels were measured using commercially available radioimmmmunoassay kits. Fur thermore atrazine, DIA, DEA, and DACT plasma levels were also measured. Statistically significant increases in ACTH levels were first detected 5 minutes after atrazine exposure at doses ≥ 100 mg/kg. Maximal increases in this hormone (↑5.6

- fold) were ob served 15 minutes after exposure to 200 mg/kg atrazine. The time

- to

- effect profile for corticosterone and progesterone increases were slightly slower with first

10 This review will only discuss data for a trazine, DIA, DEA, and DACTPage 64 of 210 statistical significant changes noted15 minutes after exposure at 50 mg/kg and above. For bot h of these hormones, maximum plasma levels were seen 30 minutes after exposure ( ↑7.3

- and 4.5 fold for corticosterone and progesterone, respectively). Exposure to DIA at doses ≥ 40 mg/kg led to increases in ACTH, corticosterone, and progesterone within 1 5 minutes of exposure ( ↑6.2, 6.3, 12.6

- fold, respectively at 161 mg/kg DIA). Similarly, DEA exposures at 173 mg/kg led to a marked increase in ACTH ( ↑11.8

- fold), corticosterone ( ↑6.4

- fold), and progesterone (13.6

- fold) within 15 minutes of exposure. In c ontrast, exposure to DACT at doses as high as 137 mg/kg led to a minimal increase in these hormones ( ≤1.3

- fold).

Plasma levels of atrazine, DIA, DEA, and DACT were detected within 5 minutes of exposure. The levels of the metabolites increased with time. Five minutes after exposure to atrazine DIA was the most prevalent metabolite but by 180 minutes DACT was the predominant metabolite seen in plasma.

The authors conclude that the rapid increase in ACTH after exposure to atrazine or its chlorinated metabolites induces adrenal steroidogenesis indicative of initial effects at the hypothalamus and/or pituitary level.

Reviewers comments: This is a well

- conducted study that carefully evaluates not only the dose

- response but also the temporal

- response re lationship between atrazine, DIA, or DEA exposure and changes in hormones in the HPA axis. Elevated ACTH, corticosterone, and progesterone levels can be seen 15 minutes after a single exposure to atrazine at doses ≥ 50 mg/kg. Although the authors did n ot establish a NOEL for this study, the no effect level may be established at 5 mg/kg

– the lowest atrazine dose tested. Interestingly, though DACT is the final and most prevalent atrazine metabolite, it has little impact on corticosterone, progesterone, or ACTH levels. The relationship between these hormonal changes and the adverse reproductive health outcomes observed after atrazine exposure, however, remains unclear. To what extent or for how long do these hormones must remain elevated before adverse effects are seen is not known at this time and cannot be determined from these data.

A.3.7 Laws, SC, Stoker, TE, Ferrell, JM, Hotchkiss, MG, and Cooper RL. 2007. Effects of Altered Food Intake During Pubertal Development in Male and Female Wistar Rats. Toxicol. Sci. 100(1): 194

- 202.

Wistar rats (n=13) were underfed 10%, 20%, 30%, or 40% less than the ad libitum food consumed by controls from postnatal days (PND) 22

- 42 (females) or PND 23

- 53 (males). This time period encompasses the age of the onset of puberty in both sexes. For any given food restriction (FR) treatment group, each animal within that group was paired with a control rat of similar initial body weight. Body weight was recorded daily. The day of preputial separation (PPS) in males and vagi nal opening (VO) in females was recorded. Females were killed by decapitation on PND 41

- 42 and liver, kidney, adrenal ovary, uterus and pituitary weights were recorded. Males were killed by decapitation on PND 53

- 54, and liver, kidney, adrenal, pituitary , left testis, ventral prostate, left epididymis, seminal vesicles with coagulating gland (with fluid) werePage 65 of 210 removed and weights recorded. Thyroid and reproductive tissues were examined histologically. Serum T3, T4 and TSH, glucose, leptin and pituitary (ma les only) prolactin and LH were measured.

The terminal body weights of FR females (PND 42) and males (PND 53) were 2, 4, 12, 19% and 2, 6, 9, 19% lower than controls for the 10, 20, 30 or 40% FR groups, respectively, being statistically significant at the top two doses. For both females and males, the age of pubertal onset was not significantly different from controls for all FR groups.
In females, the thyroid hormone endpoints were not affected by any FR group associated with 2

- 19% reduction in body wei ght. In males, there was a significant decrease in T3 and T4 at the 30% and 40% FR groups associated with 9% and 19% body weight decrease, respectively. TSH was significantly decreased in the 40% restricted group. No differences in serum testosterone, LH or prolactin were noted in any of the males. Serum glucose concentrations in males were not different from controls. Female rats in the 30% and 40% FR groups had significantly decreased serum glucose when compared to controls. Mean serum leptin concentrat ions were decreased in the 30% and 40% FR males, but were only reduced in the 40% FR females.

The absolute weights, but not the relative weights, for pituitary, adrenals, liver and kidney were significantly lower in the females with a 12% reduction in bod y weight (30% FR) and significantly lower in males with a 6 or 9% reduction in body weight (20% and 30% FR, respectively). The exception was a significant change in relative liver weight in the animals with a 6, 9, and19% body weight reduction. No histopat hological changes were observed in any organs.

In females, the absolute weights, but not the relative weights, for ovaries were decreased in the 30% and 40% FR groups. The mean uterine weights were not different from control for any FR group. In males, t he absolute, but not relative weights, for ventral prostate and seminal vesicles were significantly lower than the controls in the 40% FR.

Reviewer’s Comments: This study clarifies the relationship between altered growth and reproductive development in t he male and female rat and can be useful in interpreting effects of reproductive toxicants and endocrine disrupting chemicals. Previous studies used must greater weight loss paradigms during the postnatal period which usually resulted in over 50% weight l oss. This study uses milder food restriction limits which show that a 2 to 19% difference in weight do not interfere with the endpoints measured in the male and pubertal protocols. These observations support the concept that a 10% reduction in body weigh t is a reasonable basis for setting the maximum dose for the female pubertal protocol in conjunction with any other information concerning the toxicity of the test compound (e.g., clinical signs, other toxicity).

A.3.8 Maravilla

- Galvan, R., Fierro, R., Go nzáles

- Marquez, H. Gómez

- Arroyo, S., Jiménez, Ir and Betancourt, M. 2009. Effects of Atrazine andPage 66 of 210 Fenoxaprop

- Ethyl on Capacitation and the Acrosomal Reaction in Boar Sperm. International Journal of Toxicology. Volume 2, Number 1. pp. 24

- 32.

The purpose of this study was to evaluate the in vitro effects of atrazine and fenoxaprop

- ethyl on capacitation, spontaneous acrosome reaction (SAR) and progesterone

- induced acrosome reaction (PIAR) in boar sperm. 11

Sperm samples were obtained from the sp erm

- rich ejaculated fraction of 3 healthy fertile boars. Sperm viability was evaluated by eosin

- nigrosin staining. Only ejaculates showing more than 85% progressive motility, at least 90% viability, and less than 10% abnormality were used. Sperm capacitati on was done in TALP

- HEPES media for 4 hours. Capacitation and SAR were evaluated immediately; PIAR was evaluated 30 minutes later. The acrosome reaction (AR) was induced by adding progesterone at a final concentration of 10 μg/mL and incubating for 30 minu tes. The spontaneous acrosomal reaction without progesterone induction is called SAR. Sperm membrane status was assessed by chlortetracycline staining and analyzed with a UV epifluorescence microscope. Sperm with a uniformly distributed fluorescence over t he whole head and an intact acrosome were considered noncapacitated; sperm showing bright fluorescence in the acrosomal region beside a fluorescence

- free band in the postacrosomal region with an intact acrosome were considered capacitated; sperm with a dul l fluorescence over the whole head except for a thin, bright ban in the equatorial segment were considered acrosome

- reacted. At least 200 sperm were analyzed per slide.

Cytotoxicity was determined by incubating sperm using concentrations of atrazine (tec hnical grade, purity not reported) of 0 (control), 20, 40, 60 and 100 μM. Treatment with atrazine resulted in a dose

- related decrease in sperm viability. For 0, 20, 40, 60 and 100 μM atrazine the percent viability was 80, 60, 37, 27, and 6, respectively. T he median lethal concentrations of atrazine were calculated. Final concentrations of 8, 20, and 40 μM atrazine were used in the experiments. These concentrations corresponded to the LC 50/5 , LC 50/2 , and LC 50 , respectively. The concentrations used were suble thal for sperm in vitro , using the LC50 as a maximum. For the capacitation and PIAR studies, the herbicide was added at the beginning of the 4 hour incubation. The control was the same solvent with the herbicide (0 μM).

Sperm Capacitation: In the presence of atrazine during capacitation and PIAR, the viability of capacitated sperm decreased in a linear fashion for the 8, 20 and 40 μM dose groups. Capacitation decreased significantly only at the high dose (40 μM), to 79% of controls (p<0.05). SAR increase d 1.9 fold for the 8 and 20 μM and 2.2 fold for the 40 μM dose groups, all at p<0.05. The noncapacitated population was reduced at all concentrations, compared to controls (p<0.05).

PIAR: SAR observed in control sperm immediately after capacitation was 17%. After 30 minutes of progesterone induction of acrosome reaction, PIAR increased to 30% in

11 In this evaluation, only the results for atrazine will be discussed.Page 67 of 210 controls. This increase shows that progesterone acts as an AR inducer. All atrazine

conce ntrations produced a significant decrease (p<0.05) in noncapacitated and capacitated sperm, compared to controls. PIAR was statistically increased at 8 μM (1.6 fold), 20 μM (1.8 fold), and 40 μM (1.8 fold), compared to controls.

Similar to a previous st udy by this group (Betancourt et al, 2006), the authors review literature on atrazine effects and use, but fail to provide a sound rationale for examining direct in vitro effects on sperm. There is no literature to imply that atrazine selectively targets mature sperm and impairs their fertilizing ability after in vivo exposure. They state that “it is necessary to carry out in vitro studies to clarify the mechanisms of action of toxic compounds,” but do not provide justification for this statement. Rather they state that because atrazine and FE are used in Mexico and other countries, the aim of the study is to evaluate in vitro effects....on boar sperm capacitation and the progesterone

- induced acrosome reaction. These two statements are unrelated and do

no t provide a sound rationale for the study. There is also no evidence that atrazine reaches maturing sperm in the testis, epididymis or female tract at concentrations resembling those selected for testing in vitro

. Reviewer’s Comments: In summary, these d ata do not appear relevant to assessment of risk of atrazine after in vivo exposures nor provide biological plausibility for suspecting that atrazine may selectively target sperm and thereby impact fertility or sperm function. The authors did note that th e atrazine concentrations used in this study are not in the relevant range to produce in vivo effects.

A.3.9 Mu, H. , Shang, P. and Xu, J. (2008). Testicular Toxicity and Mechanisms of Chlorotoluron Compounds in the Mouse. Toxicology Mechanisms and Method s. 18: 399

- 403.

This study looked at the effects of chlorotoluron and atrazine ingestion on male mouse testes during the peripubertal period. 12

Groups of 20 male mice (Chinese Kunming strain) were given either chlorotoluron alone (625, 1250, 2500, 5000 mg/kg), atrazine alone (218.75, 437.5, 875, or 1750 mg/kg), or a combination of these chemicals (combination 1: atrazine/chlorotoluron: 218.75/625, 218.75/1250, 218.75/2500, or 218.75/5000 mg/kg; combination 2: atrazine/chlorotoluron: 437.5/625, 437.5/125 0, 437.5/2500, 437.5/5000 mg/kg.) There was one vehicle control group. The animals were dosed by daily gavage from day 27 to day 52. On day 52, 24 hours after the last gavage, body weights were measured and recorded and blood samples were taken. Following sacrifice testis weights were measured and recorded. Testes tissue was processed for examination by light microscope and transmission electron microscope. Superoxide dismutase (SOD) activity was measured. SOD is an antioxidant (removes superoxide radicals ).

There was a dose related decrease in relative testes weights at all dose levels for atrazine (4.6

- 13%) and combined atrazine/chlorotoluene (6.9

- 14%), although the

12 In this evaluation, only the results for atrazine will be discussed.Page 68 of 210 decreases were not statistically significant. The SOD enzyme activities increased in a do se

- related manner in animals treated with atrazine and combined, with the effects of treatment with the combination of herbicides being greater than the single chemical. Control values were not reported.

Histologic changes in the testes that occurred in t reated animals included shedding of spermatogenic cells, increased intercellular space, appearance of mild interstitial edema, and modest congestion of tissue. No tables were provided and it wasn’t clear whether these findings were for atrazine or chlorto luron, or both . Findings for mice treated with a combination of chlorotoluon and atrazine included seminiferous tubules that were shrunken and had indentations and increased shedding of spermatogenic cells and Sertoli cells. Other effects seen by electron microscopy included decreased smooth endoplasmic reticulum and Golgi (at combined dose of 625 mg/kg chlorotoluron plus 218.75 mg/kg atrazine) and heterochromatin in the mural cells which appeared in a blocked state, decreased nuclear matrix, spermatogenic cells with swollen appearance, segmental degeneration of cell membranes and tight junctions which were partially damaged (at combined dose of 1250 mg/kg chlorotoluron plus 437.5 mg/kg atrazine).

Comments: This study was poorly written such that it was un clear whether the findings were for atrazine or chlorotoluron. Consequently, this study was not useful for risk assessment.

A.3.10 Pogrmic K, Fa S, Dakic V, Kaisarevic S, and Kovacevic R. 2009. Atrazine Oral Exposure of Peripubertal Male Rats Downregu lates Steroidogenesis Gene Expression in Leydig Cells. Toxicological Sciences 111(1):189

- 197.

The purpose of this study was to attempt to identify the possible mechanism of inhibition of Leydig cell steroiodogenesis in peripubertal male Wistar rats after in vivo exposure to atrazine. The study was based on earlier published papers (described above) that identified a decreased testosterone following atrazine exposure when dosing began shortly after weaning until after sexual maturation (defined as PPS).

Male Wistar rats were treated by gavage with 0, 50 or 200 mg/kg/day atrazine (98% purity) from PND 23

- 50. Animals were sacrificed 24 hours after last dose of atrazine.

They reported that oral administration of atrazine (200 mg/kg/ay) produced significant reduction in serum T/DHT(assay they used was sensitive to both). At 200 mg/kg/day, body weight was also reduced significantly as were the weights of androgen

- dependent organs (testes and seminal vesicles at both, dorsal and ventral prostate at high dose

o nly). But LH was not altered at 50 and not significantly reduced in the 200 mg group. 50 mg/kg atrazine did not alter serum hormone levels nor the weight of androgen dependent tissues. Adrenal and Testes weights were affected in both dose groups. They pointed out that Trentacoste et al. (2001) suggested that testosterone

- reducing ability of atrazine is secondary to weight loss.Page 69 of 210 Their in vitro dosing is of interest as both doses caused significant decrease in basal and hCG stimulated testosterone release. There was no dose response and no variance presented in all the cell

- stimulation. However, the variance was presented for the dose of 10 μM hCG in a bar graph for hCG 10 μM. In addition to a decrease in T production, there was also a dose dependent decrease in the cAMP concentrations. When the cells were incubated in the presence of hydroxycholesterol, pregnolone, progesterone and androstei nedione, in every case, the amount of T produced from the Leydig cells obtained from the 50 and 200 mg/kg/ atrazine

- treated animals was reduced compared to controls. Importantly, there was no dose response. Finally the authors reported reduced levels of transcripts for CYP17A1 and 17bHSD, which seem to indicate an effect upstream and downstream of androgen synthesis. LH did appear reduced at 200 mg/kg but was not significant. This is in contrast to the observation of Friedmann, 2002 who reported that in vitro treatment with atrazine suppressed T from Leydig cells of untreated animals, but the suppression could be overcome by co

incubating with hydroxycholesterol. If these observations are both correct then there is a difference in the ex vivo vs. the in vitro exposed Leydig cell.

This group also measured the transcription of several genes involved in steroidogenesis (including the LH receptor) after p.o. administration. They report a clear dose

dependent decrease in gene expression of LHR, SR

- B1, StAR , TSPO, SF

- 1, PDE4B, 3bHSD, CYP17A1, 17bHSD in Leydig cells. There was no effect on CYP11A1 and ß

HSD. They conclude that the inhibition of all these genes following atrazine may be related to decreased c

- AMP stimulation. However, there are many inconsi stencies with the literature ( i.e. , others found SF1 increased after atrazine exposure in vitro ) and within their own data ( i.e. , cAMP is lowered, as is PDE4B....the enzyme that degrades cAMP).

Reviewer’s Comments: The quality of this manuscript is hard t o judge. There is a lot of data, much of which appears solid, however, the lack of a dose response in the Leydig cell incubation studies and the decrease in the expression of so many genes in the RT

PCR studies demands confirmation. Finally, as to whethe r or not these data support the HPG MOA or indicate a direct action of Atrazine on the Leydig cell can not be determined. In addition, it should be noted that the doses used in this study were relatively high compared to the doses eliciting the neuroendoc rine effects and used for regulatory purposes.

A.3.11 Rayner J L., Enoch R R, et al. 2007. Atrazine

- induced reproductive tract alterations after transplacental and/or lactational exposure in male Long

- Evans rats. Tox Appl Pharmacol: 218: 238

– 248.

The authors note that previous studies have shown that early postnatal exposure to atrazine delayed preputial separation (PPS) and increased incidence of prostate inflammation in adult Wistar rats. The authors stated that a cross

- fostering paradigm

was us ed in their study to determine if gestational exposure to atrazine would also result in altered puberty and reproductive tissue effects in the male rat. Timed

- pregnant Long

-Page 70 of 210 Evans rats were dosed by gavage on gestational days (GD) 15

– 19 with 100 mg/kg/da y atrazine or 1% methylcellulose (controls). On postnatal day (PND) 1, half litters were

cross

- fostered to create the following four treatment groups: birth dam or gestational exposure only (ATR

- C); milk source or lactational exposure only (C

- ATR), expos ure both pre

- and postnatal (ATR

- ATR), or neither (C

- C). Beginning on PND 37, male offspring (n>18males/group) were evaluated for puberty ( i.e. , PPS). Necropsy was performed on either PND120 (n = 10/group) or 220 (n = 9

- 10/group); organs included pituitar y gland, testes, lateral and ventral prostates, and seminal vesicles. Serum prolactin, testosterone, androstenedione and estrone levels were measured by radioimmunoassay. The left lateral prostates were analyzed for myeloperoxidase activity. Histologica l examination of the right lateral and ventral prostates were conducted. The distribution scoring and severity classification were provided in the study. Body weights were monitored throughout the study.

Mean body weight of the ATR dams on GD19 was red uced 13% when compared to control dams. The male offspring in the ATR

- ATR group were 6.5% smaller when compared to offspring in the C

- C and C

- ATR groups on PND4. A significant 2 1⁄2 day pubertal delay was seen only in males in the ATR

- ATR group. Body wei ghts at the time of PPS were also decreased in the ATR

- ATR animals.

At PND120, the lateral prostates of the ATR

- ATR offspring group weighed significantly more than those in any other group, even when corrected for body weight. The average ATR

- ATR later al prostate weight was 49% higher than control on PND120. At PND220, lateral prostate weights were significantly increased (45

– 110%) in animals within all ATR exposure groups compared to control animals. When adjusted for body weight, animals in ATR

- C and ATR

- ATR groups had significantly increased lateral prostate weights.

At PND120, necropsy evaluation of offspring indicated macroscopic abnormalities of the left and right lateral prostate in the ATR

- ATR group; the abnormalities were characterized by nodular pale white foci present on the outer surface. At PND 220, all atrazine groups had the abnormalities, mostly on the right lateral prostate.

Histological examination of lateral and ventral prostates revealed an increased incidence and distribution of inflammation in the lateral prostates of C

- ATR males at PND 120. Only one out of ten ATR

- ATR males also exhibited inflammation; there was no inflammation in the C

- C and ATR

- C groups. The C

- ATR and ATR

- ATR lateral prostates had neutrophils as the pred ominant inflammatory cells. In regard to ventral prostates at PND 120, inflammation was evident in all groups (C

- C, ATR

- C, C

- ATR and ATR

- ATR) indicating no effect of treatment. The authors stated that it is difficult to assess inflammation in the ventral prostate because of a high background present. The authors also noted that although necropsy revealed macroscopic abnormalities on the surface of the lateral prostate of males in the ATR

- ATR group, these abnormalities “may not have penetrated deep within the gland.” By PND220, lateral prostate weights were significantly increased for ATR

- C and ATR

- ATR groups, but there were no significant changes in inflammation in either the lateral or ventral prostate.Page 71 of 210 The results of the myeloperoxidase assay demonst rated no significant effects; the authors noted the discrepancy between the assay and the pathological findings. The authors explained the discrepancy as possibly due to not having both lateral prostates for assay (one used for the assay, the other for pa thology). No statistically significant differences in serum hormone concentrations of total testosterone, androstenedione, estrone, and prolactin were observed among the different treatment groups.

The authors concluded that the results of the study su ggest that the developing lateral prostate gland in the Long

- Evans rat is sensitive to atrazine

- induced changes that are transplacental or via nursing. In addition, lactational exposure to these atrazine

- induced changes increases lateral prostate inflamma tion.

Reviewer’s Comments: This study only examines one dose, so there are no dose response evaluations. The authors do not mention the time of day which they killed the males at 120 or 200 days of age or how long the necropsy lasted, as this would be very important when evaluating effects on the endocrine system. The increased lateral prostate weights are reflective of an increase in lateral prostate inflammation, which has been found previously following early postnatal exposure of atrazine to the l actating dam (Stoker et al., 1999). There is also a delay of PPS in the ATR

- ATR dose group only, indicating that it is the combination of gestational exposure to the dam and the postnatal milk from those mothers responsible for the effect. Rosenberg et al, 2008 also observed a delay in PPS at 100 mg/kg with an oral perinatal exposure only, a condition that would be similar to the ATR

- CON group in whom Rayner found no effect on PPS. It should be noted that these effects in the Rayner et al. study occur at doses higher than those eliciting disruption of the LH surge.

A.3.12 Rosenberg, B.G., Chen, H., Folmer, J., Liu, J., Papadopoulos, V., and Zirkin, B.R. (2008). Gestational Exposure to Atrazine: Effects on the Postnatal Development of Male Offspring. J. Andrology 29(3):304

- 311.

In this study, the authors investigated the impact of atrazine exposure during gestation on the development of Sprague

- Dawley (SD) rats male offspring. Fourteen to 16 pregnant dams were exposed via gavage from gestation day ( GD) 14

- 21 to 0, 1, 10, 50, 75, or 100 mg/kg/day of atrazine (purity 98.5%). The male offspring of 7

- 8 dams per dose group were sacrificed on post

- natal day (PND) 0. The testes from these males were collected and used to measure testicular testosterone l evels in the newborn pups. The litters of the remaining 7

- 8 dams were culled on PND2 to 8 pups (4/sex/litter). Body weights and food consumption of pregnant dams were recorded daily from GD 14

- 21. On PND21 the body weights and anogenital distance (AGD) we re measured for each pup. The 4 male pups from each litter were also examined for preputial separation beginning on PND35. On PND 60 the pups were sacrificed by decapitation and their blood (serum), testes, ventral prostate, and seminal vesicles collected . The testes, ventral prostate, and seminal vesicles were weighed. Testosterone levels were measured in both the serum and intratesticular fluid.Page 72 of 210 Maternal body weight was statistically significantly reduced at doses ≥ 75 mg/kg/day starting on GD18. This decrease coincided with a 33

- 47% decrease in food consumption for the 50, 75 and 100 mg/kg/day dose groups. Litter size was unaffected by atrazine treatment. However, pup death between PND 0 and 2 was increased by approximately 10

- 25% at doses ≥ 10 mg/kg/day, with statistical significance attained at the 75 and 100 mg/kg/day doses (~20

- 25%). Pup weight decreases of 10

- 17% were reported for pups whose dams were exposed to doses 50 mg/kg/day or higher. None of these pup weight decrements, however, we re statistically significant.

Intratesticular testosterone levels measured from neonates did not reveal any statistically significant effects at any dose level. Although a 60% increase in intratesticular testosterone was noted at the 10 mg/kg/day dose le vel, there was no statistical significance or dose response for this effect ( i.e., testosterone levels at ≥ 50 mg/kg/day were comparable to control). It is noteworthy, that at this dose level a non

statistically significant increase in AGD was observed in males on PND 21. while AGD was significantly reduced at 100 mg/kg/dose levels on PND 21.

A statistically significant delay in PPS ranging from 1

- 3 days was noted at 50 mg/kg/day and above. (Note: Graph and text differ: graph indicates ss only at 100 m g/kg/day (p<0.004) while text says ss at ≥ 50 mg/kg/day (p<0.05, p<0.01)).

On PND 60, the animals used to assess PPS and AGD were sacrificed and their testes, seminal vesicles, and ventral prostate weights measured. In addition, testosterone levels in b oth the serum and intratesticular fluids were measured. No changes in terminal body weight or organ weights were noted. Serum testosterone levels, however, were decreased at ≥ 50 mg/kg/day while intratesticular testosterone was reduced at doses of 75 and 100 mg/kg/day. [Note: graph and text report of T differ; they are reversed.]

The authors of this study concluded that in utero exposure resulted in changes that manifested in the neonatal, peripubertal, and adult life stages. The authors, however, did point out that these effects were observed at concentrations that “are unlikely to be experienced under any but experimental conditions.”

Reviewer’s Comments : The highest two doses 75 and 100 mg/kg/day resulted in significant reductions in the dam’s BW gain. There was a dose

- dependent loss of pups

-- ~20

- 25% pup death at 75 and 100 mg/kg from PND 0

- 2. There were no differences in neonatal T at PND 0, but ther e did appear to be effects on androgen dependent measures later in life, including reduced AGD at PND 21, a delay in PPS in the 100 mg/kg group and a decrease in serum T at 60 days of age in the 50, 75 and 100 mg/kg group. Oddly, none of the androgen

- depe ndent tissue weights, including the seminal vesicles or ventral prostate were affected even at the highest dose. It should be noted that the effects on maturation and altered androgen status occurred in this study at dose levels substantially higher than those leading to LH surge disruption, which serves as the basis of the Agency’s current risk assessment.Page 73 of 210 A.3.13 Stoker TE, Guidici DL, Laws SC, Cooper RL. 2002. The Effects of Atrazine Metabolites on Puberty and Thyroid Function in the Male Wistar Rat. T oxicological Sciences 67: 198

- 206.

Male Wistar rats were gavaged (between 8 and 9 am) from PND 23

– PND 53 with atrazine metabolites (97

- 98% purity), deethylatrazine (DEA), deisopropylatrazine (DIA), and diaminochlorotriazine (DACT) at doses equivalent to the atrazine equimolar doses (AED) of 0, 6.25, 12.5, 25, 50, 100, or 200 mg/kg/day.. The day of preputial separation (PPS) was measured. Males were killed by decapitation 2 hours following the last dose on PND 53. The pituitary, adrenal, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were weighed and examined histologically. Blood and serum were assayed for hormones (testosterone, estrogen, LH, prolactin, TSH, T4, T3).

There was a significant decrease in body weight se en with all the metabolites at the two highest dose groups. For all metabolites at 200 mg/kg/day, there was a greater than 10% decrease in body weight at PND 43 and 53. For all metabolites at 100 mg/kg/day, body weight decreases ranged from 9

- 12% at PND 4 3 to 7

- 13% at PND 53, with DEA being the metabolite exceeding 10% body weight decrease at both time points.

The day of PPS was delayed by all 3 metabolites. For DIA, PPS was significantly delayed at 25, 100, and 200 mg/kg/day AED by 1.2, 1.2 and 3.4 days , respectively, resulting in a NOAEL for delayed PPS of 12.5 mg/kg/day. For DEA, PPS was significantly delayed at 25, 100, and 200 mg/kg/day AED by 1, 2 and 4 days, respectively, with a NOAEL of 12.5 mg/kg/day. For DACT, PPS was significantly delayed at 12.5, 25, 100, and 200 mg/kg/day AED by 1, 2.3, 1, 4, and 4.2 days, respectively with a NOAEL of 6.25 mg/kg/day.

Testicular weights were similar to controls in all treatment groups for all three metabolites. When males were killed on PND 53, DEA (100 and 200), DIA (50

- 200 AED), and DACT (200 AED) treatments caused significant reduction in ventral prostate weight, while the highest doses of DIA and DEA resulted in significant decrease in lateral prostate weight. Seminal vesicle weight was reduced at 100 an d 200 AED by all three metabolites. Epididymal weights were decreased in the DEA (200 AED), DIA (200 AED), and DACT (100 and 200 AED) treatment groups.

Serum testosterone was significantly decreased only in DIA exposed males at 100 and 200 AED. Serum est rone was significantly increased only by DACT at 100 and 200 AED.

The mean anterior pituitary weight was significantly decreased on PND 53 at 100 and 200 AED by DEA and DACT and the highest dose of DIA. On PND 53, serum LH showed a significant trend for a dose

- dependent decrease from control to high dose in the DIA group. Pituitary LH was significantly decreased in the 100 AEd of DIA and the 200 AED of DEA compared to controls. Pituitary prolactin content was significantly decreased at 100 and 200 AEd of DEA and at 200 AED of DACT.Page 74 of 210 TSH, T3 and T4 were unaffected by any treatment group. No effects were observed on thyroid histology of high dose or controls.

Minimal hypospermia (defined as rats in which the overall density of spermatozoa within the epidid ymal ducts appeared to be decreased compared to control samples() was seen in 25% of the high dose group of DIA, 40% of the high dose group of DACT, and 12.5% of the high dose group of DEA. The author reported that this observation is probably a result of the length of the delay in puberty in the high dose groups of atrazine metabolites and the age of sacrifice.

Reviewer’s Comments: This study shows the effects of the three primary atrazine metabolites on pubertal onset and growth of androgen dependent tis sues. Interestingly, many of the same effects were observed including delayed PPS and decreased reproductive tract development. DIA was the only metabolite which resulted in decreased serum testosterone and DACT increased serum estrone. The doses appear to be similar to those that were effective with atrazine for the same endpoints, including PPS. DACT delayed puberty at the same doses (12.5 to 200 AED). It is noteworthy that the lowest NOAEL for the effects reported by Stoker et al. 2002 (6.25 mg/kg/d ay in atrazine molar equivalents) was higher than the NOAEL (1.8 mg/kg/day) for attenuation of the LH surge currently used as the point of departure in the Agency’s risk assessment.

A.3.14 Stoker, T.E., Laws, S.C., Guidici, D., and Cooper, R.L. 2000. Th e effects of atrazine on puberty in male wistar rats: An evaluation of a protocol for the assessment of pubertal development and thyroid function. Toxicological Sciences. 58: 50

- 59.

Intact male Wistar rats (n=6

- 20) were treated with atrazine (97.1% pur ity) via gavage at doses of 0, 12.5, 25, 50, 100, 150, or 200 mg/kg/day from postnatal day (PND) 23 to 53. An additional pair

- fed group was added to compare effects of any decreased food consumption in the high dose group. Daily body weight and preputial separation were measured. Males were killed by decapitation on either PND 45, 53, (or 54) or 120. The pituitary, thyroid, testes, epididymides, seminal vesicles, and ventral and lateral prostates were weighed and examined histopathologically. Blood and serum were assayed for hormones (testosterone, estrogen, LH, prolactin, TSH, T4, T3).

There was a significant reduction in body weight in the high dose (200 mg/kg/day) on PND 43 (14%) and PND 50 (17%). The pair fed group of 200 mg/kg/day had a significant reduction in body weight on PPS day (13%), PND 43 (20%) and PND 53 (15%).

Preputial separation was significantly delayed in the 12.5, 50, 100, 150, and 200 mg/kg/day dose groups by 2.3, 1.8, 1.7, 1.6 and 3 days as compared to controls. PPS was also dela yed by 2 days in the pair

- fed animals compared to controls. The delay inPage 75 of 210 PPS observed in the pair

- fed animals was not as severe as that observed in the atrazine 200 mg/kg/day group.

This study showed that atrazine suppressed the growth of androgen depend ent reproductive tissues at 50 mg/kg and up (ventral prostate). Ventral prostate weight was also decreased in the pair

- fed group. Seminal vesicle and epididymal weights on PND 53 were decreased in the 200 mg/kg/day atrazine group and pair

- fed animals. By PND 120, all reproductive tract organ weights returned to control levels in all treatment groups, except for the ventral prostrate which was still significantly decreased in the 200 mg/kg/day atrazine group (784.8 **±** 46.1, controls vs 638.13 **±** 25.3, 250 mg/kg/ day). Lateral prostate and testicular weights were not different from control in any of the atrazine or pair

- fed rats killed on PND 53.

There was no effect on serum testosterone at 53 days of age, but intra

- testicular testosterone was decreased on PND 45 which might explain the delay in pubertal development. There was no significant effect on serum LH on PND 53, but serum estrogens were increased at 200 mg/kg/day. These effects did not appear to be due solely to decreased growth, as there were effects a t doses which did not affect growth (both PPS and tissue weights).

Reviewer’s Comments: Peripubertal exposure to atrazine resulted in delayed puberty onset and delayed growth of the reproductive tract of male rats. These data support the reported alterati on of the hypothalamic control of pubertal progression.

A.3.15 Trentacoste, S V.. Friedmann, AS, Youker, RT, Breckenridge, CB, and Zirkin, BR. 2001. Atrazine Effects on Testosterone Levels and Androgen

- Dependent Reproductive Organs in Peripubertal Ma le Rats. Journal of Andrology, Vol. 22, No. 1, January/February 2001 ; pp. 142

- 148.

Male Sprague

- Dawley rats received atrazine (96.1%) from PND 22

- 47 by gavage in methyl cellulose at doses of 0, 1, 2.5, 5, 10, 25, 50, 100 or 200 mg/kg/day (n = 9

- 12 rats/d ose) and killed by decapitation on PND 48 (24 hours following last dose; doesn’t state time of day killed). A food restriction study was conducted with rats pair fed to the 100 mg/kg/day atrazine treated group. In life measures included daily (?) body wei ghts. The day of preputial separation (PPS) was measured in controls and only the 100 and 200 mg/kg/day dose groups. Testosterone (serum and interstitial fluid) and serum LH concentrations were measured.

Body weight (9%, 100 mg/kg/day/21%, 200 mg/kg/day), seminal vesicle (32%/55%) and ventral prostate (26%/48%) weights, serum T (34%/32%) and intratesticular fluid T (41%, 45%), LH (17%, not s.s./20%) and PPS (3 days/4 days) were significantly reduced in the 100 and 200 mg/kg/day groups. PPS was only evaluated in the 100 and 200 dose groups. As body weight was affected by the atrazine 100 and 200 mg/kg/day, a group of males were pair fed to the 100 mg/kg/day group. The underfed only animals had a decrease in T and LH on PND 47 that was similar to the 100 mg/kg/dose group.Page 76 of 210 The authors concluded that these results indicate that atrazine exposure, up to 50 mg/kg/day, had no effect on the hypothalamic

- pituitary

- testicular axis, and that at levels greater than 50 mg/kg per day, the effects observed in response to atrazine could not be distinguished from the effects observed after reduced food consumption. The author’s emphasized that caution must be exercised before concluding that atrazine (or any potentially toxic chemical) has direct, detrimental effects when the toxicant also produces significant changes in body weight. They further emphasize that body weight must be both monitored and accounted for in the assessment of potential toxic effects.

Reviewer ’s Comments: The study author’s contention that the changes in the male reproductive tissue weight and PPS changes observed was the result of decrease body weight is not supported by an earlier study by Stoker et al., 2000. In that study, Stoker examined s imilar measures in the male Wistar rat. This group reported that 100 mg/kg/day reduced BW approximately 7% (vs. approximately 10% in the present study with Sprague

- Dawley males). However, Stoker also found a decrease in ventral prostate weights at 50 mg/k g/day males (which had BW reduction of 3%) and delays in preputial separation following 12.5 mg/kg/day and above. Stoker also noted lower serum testosterone values in males treated with 25 mg/kg/day and above, which was very similar to that observed by Ro senberg et al., 2008, following gestational dosing). Stoker et al., also pair fed males during the experimental period base on the food consumed by the 200 mg/kg/day animals. The body weight in the pair fed animals was 84% of control body weights (reduc ed 16%). Serum T was not reduced in Stoker et al study, however they did have smaller ventral prostates, seminal vesicles and a delay in PPS (albeit significantly less than that observed in the 200 mg/kg/day rats). Importantly, the Trentacoste et al., pa per did not look at the onset of preputial separation in animals dosed with less than 100 and 200 mg/kg/day, the very endpoint that set the LOAEL an NOAEL in the Stoker paper. No explanation is given for this omission. Freidman (2002) reported a signific ant decrease in both serum testosterone and interstitial testosterone in Sprague

- Dawley rats treated during the same PND period, and body weight was reduced 7% (230 = control 215 = atrazine 50 mg/kg/day from 22

- 48.

A.3.16 Victor

- Costa, AB, Bandeira SMC, Oliveira AG, Mahecha GAB, Oliveira, CA. 2010. Changes in Testicular Morphology and Steroidogenesis in Adult Rats Exposed to Atrazine. Reproductive Toxicology. oi10.1016/j.reprotox.2009.12.006.

This study evaluated the effects of atrazine on the steroid ogenic pathway in adult male rats by analyzing the 3 **β**

- HSD expression, plasma and testicular estrogen and testosterone levels, expression of androgen receptor, and morphophysiological changes in the testes.

Adult male Wistar rats (4

- 7/group) (120 days old) were given daily doses of atrazine (purity not provided) via gavage for 7 days (300 mg/kg/day), 15 days (50 and 200 mg/kg/day) or 40 days (200 mg/kg/day). A commercialized mix of atrazine called Gesaprim ® was used. This formulation consisted of 500 g/l at razine and 500 g/l of inert ingredients. Control animals were treated for similar periods with the vehicle, corn oil.Page 77 of 210 After treatment, rats were weighed and sacrificed. Testes and adrenal glands were weighed. Testes were examined histopathologically. Cha nges in the expression of 3 **β**

hydroxysteroid dehydrogenase (3 **β**

- HSD) enzyme and androgen receptor (AR) in the testes were studied by immunohistochemistry. 3 **β**

- HSD is the enzyme responsible for various stages of the steroidogenic pathway, including synthesis of adrostenedione, the testosterone precursor. Adrenal glands were also evaluated for the 3 **β**

- HSD by immunodetection for comparison between steroidogenic tissues. Immunostaining was quantified by computer

- assisted image analysis. AR staining was quantified in 30 interstitial Leydig cells as well as 30 Sertoli cells lining seminiferous epithelium stages VII or VIII, per animal, except for those treated with atrazine 200 mg/kg/day/40 days in which the testes were atrophic and the stages of the seminiferous epi thelium cycle could not be identified. In this case, the Sertoli cell positivity was measured in randomly observed tubules. Plasma testosterone and estradiol were measured by both radioimmunoassay (RIA) and enzyme linked immuno sorbent assay (ELISA). Weste rn blot assays were performed to determine the specificity of the antibodies and the changes in the expression of 3 **β**

- HSD enzyme in testis and adrenal and androgen receptor in testes of treated groups.

There were significant body weight decreases (p<0.05) in the rats treated with atrazine 200 mg/kg/day (15 and 40 days) (35%) and 300 mg/kg/day (7 days) (38%). A significant increase (p<0.05) in the relative and absolute testes weights were seen in rats treated with atrazine 200 mg/kg/day/15 days (80% and 16% ) and 300 mg/kg/day/7 days (83% and 14%). However, exposure to atrazine 200 mg/kg/day/40 days resulted in a 41% and 60% reduction in the relative and absolute testis weight, respectively. There was a significant increase (p<0.05) in the relative adrenal we ight of 64%, 104%, and 41% for the rats treated with atrazine 200 mg/kg/day/15 days, 200 mg/kg/day/40 days, and 300 mg/kg/day/7 days, respectively. There was no effect on body weight, testis or adrenal weights in animals treated with atrazine 50 mg/kg/day for 15 days.

Plasma testosterone levels, as measured in RIA assays, were statistically decreased (p<0.05) compared to controls in all treated animals (65% for 50 mg/kg/day/15 days; 85% for 200 mg/kg/day/15 days; 86% for 200 mg/kg/day/40 days; 87% for 300 mg/kg/day/7 days). ELISA assay confirmed the reduction in testosterone (data not provided). Testicular levels of testosterone were also significantly (p<0.05) reduced (32%) after treatment with atrazine 200 mg/kg/day/15 days. However, plasma and testic ular estradiol levels were significantly increased by 33% and 38%, respectively, after treatment with atrazine 200 mg/kg/day/15 days. At all other dose levels, the plasma and testicular estradiol levels were similar to controls.

Atrazine exposure caused changes in testis morphology. Atrazine exposure caused dilation of seminipherous tubules at doses of 200 mg/kg/day/15 days and 300 mg/kg/day/7 days. Pleomorphic Leydig cells with irregular nuclei were observed in all atrazine treated animals. Atrazine 200 mg/kg/day/40 days resulted in testis atrophy and reduction in the seminiferous tubules lumen. In this group giant multinucleated bodies and a large number of apoptotic cells were frequent within the seminiferous tubules.Page 78 of 210 Immunopositivity for AR was dete cted in Leydig cells, Sertoli cells, and Myoid cells of both treated (200 mg/kg/day/15 and 40 days; 300 mg/kg/day/7days) and control rats. There was no difference in AR protein levels when animals treated with atrazine 200 mg/kg/day/15 days and controls we re compared by Western blotting assay. Quantification of the AR immunopositivity in specific cell types after exposure to atrazine at the same dose (200 mg/kg/day/15 days) showed equal AR intensity in Sertoli cells and Leydig cells compared to controls, wh ich confirmed the Western blotting results. Treatment with higher doses of atrazine (300 mg/kg/day/7 days) resulted in decreased AR intensity in both Sertoli and Leydig cells. Longer treatment with atrazine (200 mg/kg/day/40 days) resulted in decreased AR staining in Sertoli cells but increased AR staining in Leydig cells. It was noted that the rats treated for 40 days had atrophied testis and most of the tubules presented AR

- positive Sertoli cells, with reduced staining intensity.

In control animals, 3 **β**

- HSD positivity in the testis was found in the Leydig cells cytoplasm. After atrazine exposure, there was a great reduction in Leydig cells 3 **β**

- HSD immunoreactions (either weakly stained or negative). These data were confirmed by Western blotting, in which rats treated with atrazine 200 mg/kg/day/15 days showed significant reduction (40%) in the intensity of 3 **β**

- HSD positive bands in the testis. The expression of 3 **β**

- HSD immunoreaction was found in cells of the three zones of the adrenal cortex. The pattern o f distribution and intensity of the 3 **β**

- HSD immunoreaction in the adrenal cortex was similar in both control and treated animals. Western blotting was in agreement with these results, showing that the 3 **β**

- HSD protein was not altered in adrenal cortex of trea ted animals compared to controls.

Reviewer’s Comments: This study used some unusual treatment regimens. There is no dose

- response examined, as there is only one dose per treatment period except in the 15 day exposure. For example, in the 15 day exposure , they include a 50 and 200 mg/kg dose, but do not show any effects for the 50 mg/kg except for decreased testosterone. It is difficult to interpret the results with this experimental design and the high doses. They do include some measures that other st udies in this area did not examine, including the measurement of testicular and adrenal 3B

- HSD and testicular androgen receptors. 3B

- HSD is a key enzyme in the androgen biosynthetic pathway, but changes in the expression or protein levels could be a resul t of the suppression of luteinizing hormone. The fact that they found a decrease in B

- HSD in the testes is different than the Pogrmic paper wherein this was one of only two (of the many examined) that did NOT change, but the current paper looks at the pro tein using immunocytochemistry and the Pogrmic paper looked at message. The changes in testicular weight are interesting and haven’t been shown before. For example, testis increased weight with transient exposures (200 for 15 days or 300 for 7 days), but showed atrophy with exposed for 40 days at 200 mg/kg. The larger weights correlated with increased lumens of the seminiferous tubules. The increased lumen is likely due to increased fluid accumulation (backflow). Over a period of time, this can lead to atrophy, which may be why it is observed in the 40 day exposure. The high doses and the factPage 79 of 210 that 50% of this was made from “other

- ingredients” in the commercial mixture make the results difficult to interpret. This paper adds little to what we know.

A.3.17 Fraites MJP, Narotsky MG, Best DS, Stoker TE, Davis LK, Goldman JM, Hotchkiss MG, Klinefelter GR, Kamel A, Qian Y, Podhorniak L, Cooper RL. (2011). Gestational Atrazine Exposure Effects on Male Reproductive Development and Metabolite Distributio n in the Dam, Fetus, and Neonate. Reprod. Toxicol., doi:10.1016/j.reprotox.2011.04.003

In this study the authors investigated the potential impact of in utero exposure on male reproductive development. Various androgen

- dependent endpoints were evaluated including: (i) testosterone production at birth and on PND 59, (ii) rough and tumble play behavior, (iii) AGD and PPS, and (iv) androgen

- dependent organ weight s. In addition, the investigators studied the transfer of atrazine and its metabolites into the fetus after maternal exposure during gestation (GD 18

- 20 or GD 14

- 20). 13

Time

- pregnant Sprague

- Dawley rats (12 dams/dose) were exposed to 0, 1, 5, 20, and 100 mg/kg/day atrazine (97% a.i. purity) by gavage once daily (s.i.d) or divided into 2 daily doses (b.i.d) during GD 14

- 21. At birth, 2 male pups/litter were selected for testis histology or unstimulated or LH

- stimulated testosterone production by the testis . AGD measures were taken on PND 7 and beginning on PND 38 pups were examined daily for PPS

– an indicator of puberty onset. On PND 30

- 33, male pairs were assessed for rough and tumble play behavior (pinning, chase, on the back posture, wrestling, or box ing). At the time of necropsy, 2 males/litter were decapitated within 15 seconds of removing them from the cage and their trunk blood was collected. Serum testosterone and LH levels were measured. In addition, the pituitary, testes, left epididymis, sem inal vesicles with coagulating gland (with fluid), and ventral prostate were removed and weighed.

Exposure to atrazine did not affect PPS, AGD, rough and tumble play behavior, serum or interstitial testosterone, serum LH, unstimulated and LH

- stimulated testosterone production by the testes (either at birth or on PND 59), or androgen

- dependent organ weights. The only atrazine

- related effects observed in the study occurred at the 100 mg/kg/day dose level and included decrease in maternal body weights, pup body weights ( ↓6

- 12%), and pup viability between PND 1

- 4 after s.i.d administration (9.7% pup loss vs 1.4% in control) or b.i.d. administration (33.6% pup loss vs 0.9% control). A statistically significant decrease in absolute pituitary weight (( ↓12%) wa s also observed at the 100 mg/kg/day dose level on PND 59 after s.i.d administration (but not b.i.d) but this was likely related to the decreases in pup weight since relative pituitary weight was unaffected.

Reviewers comments: This is a well conducted st udy that indicates that at the doses tested (1

- 100 mg/kg/day) late

- gestational exposure to atrazine does not affect male

13 The results of the atrazine distribution experiments were described in review A.2.1 3 in Appendix A of this document and will not be discussed further in this review

.Page 80 of 210 reproductive development or the androgen

- dependent endpoints measured in this study.

A.4 AROMATASE

Summary: Effects on Aromatase and Steroidogenesis, Andrology

Aromatase is the enzyme that catalyzes the conversion of androgens to estrogens. It is expressed in the gonadal and extragonadal tissues and is critical for maintenance of homeostasis between the sex steroids in males and fema les which is necessary for reproductive function. Although aromatase is a product of a single gene (CYP19) in mammals, its expression is quite complex as it is regulated in a species

- and tissue

dependent manner by differential uses of multiple promoters (Simpson et al. , 2002). It is well documented that environmental contaminants can inhibit the catalytic activity of this enzyme (Vinggaard et al., 2000; Santen et al., 2009). However, studies reporting an increase in the activity of aromatase are not as numerous.
Approximately ten years ago, Sanderson and his colleagues reported that atrazine induces estrone production and aromatase (CYP19) mRNA in H295R adrenal cortical carcinoma cells (Sanderson et al. 2000; 2001). These authors reported that ele vated aromatase activity, as measured using the tritiated water release assay, was observed in cells exposed to atrazine (low

- effect level, LOEL, 0.3  M) for at least 24 hours. Since these original reports, several studies using a variety of cell lines ha ve confirmed the atrazine

- dependent increases in estrogen production in H295R cells (Heneweer et al., 2004; Higley et al., 2010) and human placental carcinoma (JEG

- 3) cells (Sanderson et al., 2001; Laville et al., 2006). Finally, Holloway et al. (2008) al so reported that aromatase activity in human granulosa

- lutein primary cell cultures following treatment with atrazine (1.0 μM) was 2

- fold higher than that observed for the controls. A commonality among these studies is that the effects on estrogen product ion are only observed in the various cell types when exposed to atrazine for at least 24 hours. This finding supports the hypothesis that the effects are not mediated through a direct alteration of the catalytic activity of aromatase, but rather likely in volve gene transcription of the enzyme.

To date, the cellular mechanisms responsible for the increased production of estrogens by aromatase have not been fully characterized. Sanderson et al (2000, 2002) reported that the time

- response profile for t he induction of aromatase activity, CYP19 mRNA, and cyclic adenosine monophosphate (cAMP) in H295R cells following exposure to atrazine was similar to that of 8

- bromo

- cAMP, a known stimulant of the protein kinase

- A pathway that mediates the induction of ar omatase. These authors suggested that the atrazine

dependent induction of estrogen in H295R cells results from increased CYP19 mRNA synthesis, which may be stimulated by elevated intracellular cAMP. In contrast, Fan et al., (2007a, b) reported that atrazi ne binds directly to steroidogenic factor 1 (SF

- 1), an orphan receptor that serves as a transcription factor for the expression of CYP19 mRNA through its interaction with aromatase promoter II. While these studies focused primarily on the up

- regulation of aromatase gene expression, additional studiesPage 81 of 210 demonstrated that other cellular target sites exist for atrazine and that these target sites can impact the steroidogenic process. Suzawa and Ingraham (2008) reported that atrazine does not bind directly to t he SF

- 1, but instead stimulates three signaling pathways known to activate several of the NR5A receptors [SF

- 1,hLRH

- 1 (human liver receptor homolog), zff1d]. These authors also reported an upregulation of the expression of a group of important steroidogeni c genes to include StAR, inhibin

- α, P450scc, and11βHSD in JEG3 cells following exposure to atrazine. Thus, these findings demonstrate that atrazine can exert a much broader effect on steroidogenesis. Finally Albanito et al. (2008) reported a series of in vitro experiments designed to evaluate the mechanism of action for atrazine in BG

- 1 cells (human ovarian cells). Their data confirm that atrazine does not bind directly to the nuclear ER  as previously reported by Cooper et al. (2007). However, atrazine did not alter aromatase activity or gene expression in the BG

- 1 cultures following a 40

- hr exposure, a finding that contrasts with Sanderson et al. (2000) for the H295R cell line and may be a reflection of a constitutively high aromatase activity that pre vents further amplification by exposure to atrazine. A novel finding of this study was the stimulation of extracelluar signal

regulated kinase (ERK) phosphorylation and c

- fos in the BG

- 1 cells following exposure to atrazine (1  M). Using specific signaling inhibitors and gene silencing, these authors report that this effect is mediated through the G

- protein

- coupled receptor 30 (GPR30), a membrane bound orphan receptor that mediates rapid estrogen signals

The range of the LOELs (0.3

– 1 μM) for the observe d effects on cellular mechanisms provides a common factor among the in vitro studies. Recent studies in rats and mice have measured plasma and tissue concentrations of atrazine and metabolites following in vivo exposures (Ross et al. , 2007; Laws et al. 20 09, Fraites et al. , 2009). Although a complete profile of serum and tissue atrazine concentrations for the different dosing durations tested in vivo is not available, the data reported by Fraites et al. , for the serum concentrations of the active atrazine metabolite DIA show that it reaches the 2

- 4 μM range within 15 minutes after four daily doses of 75 mg/kg/day via gavage. Again, although more data would be needed to accurately assess the internal dose in animals treated for different durations, this da ta does indicate that the concentrations used in the in vitro studies discussed above are not excessive. However, these in vivo doses are still substantially higher than those selected for points of departure in the 2003 risk assessment.

In summary, the in vitro studies demonstrate that atrazine can alter aromatase activity and gene transcription. The more recent studies suggest that the cellular responses following atrazine exposure involve a complex array of signaling events, Importantly, the fact tha t atrazine may exert a broad influence on steroidogenesis is in accord with the in vivo studies reporting increased adrenal (Fraites et al., 2009; Laws et al., 2009) and testicular (Modic, 2004) steroidogenesis following acute chlorotriazine dosing. It is interesting to note that although changes in the production of serum steroid hormones have been identified in vivo following atrazine exposure including estrogen, albeit at high doses (Stoker et al., 2000), alterations in aromatase gene expression or acti vity have not been observed in the rat testes (Modic 2004) or mouse reproductive tract (Rivest and Sanderson, 2010). Thus, suggesting that while steroidogenesis may bePage 82 of 210 increased as a result of atrazine exposure, this event is likely not mediated by aromat ase.

Three in vitro studies have been conducted over the last seven years to evaluate the potential effect of atrazine exposure on sperm. Given the effects of atrazine on a number of hormone

- dependent events including triggering early reproductive

senesc ence in female rats and causing delays in puberty onset, it is hypothesized that atrazine may also affect sperm parameters ( e.g., motility, viability). Two of the studies used porcine sperm while the remaining study used human sperm to explore the potenti al impact of atrazine exposure on sperm. The researchers hypothesized that effects seen after in vitro exposure to atrazine could be correlated with changes in fertility or reproductive success after in vivo exposure.

In a study designed to evaluate the effect of atrazine exposure on sperm motility, semen collected from Duroc boars was subjected to a number of evaluations after exposure to 0, 50, 100, or 500 μM atrazine (Betancourt et al., 2006). Exposure to concentrations ≥ 100 μM atrazine elicited a co ncentration

- dependent and statistically significant decrease in sperm viability ( ↓8

- 21%) and progressive motility ( ↓23

- 75%). The authors speculated that the sperm motility effects seen after in vitro exposure would lead to an adverse effect on the sperm’s “fertilizing ability” and hence male reproduction. This statement is not supported by empirical data but rather conjecture based on the changes in sperm motility observed after in vitro exposures. This is but one of the numerous limitations of this stud y. The authors did not take into consideration the plausibility that atrazine would come into direct contact with sperm after in vivo exposure. Moreover, this study does not take into account that atrazine is quickly metabolized in the body and therefore the substance reaching the testes is most likely an atrazine metabolite assuming it could reach the testes. Finally, there is no attempt to correlate the in vitro concentrations tested to any in vivo concentrations that live sperm may encounter during mat uration in the testis, epididymal transit, in seminal fluid, or in the female oviduct where fertilization takes place. In conclusion, this study evaluates mature sperm in vitro and attempts to extrapolate those findings to potential effects on male reprod uctive performance. In fact the relatively high concentrations of atrazine suggests that it may have toxic properties at the level of the sperm membrane or mitochondria in vitro but shows no clear relevance to in vivo exposures or a potential MOA.

A second study conducted by the same research group evaluated the effect of atrazine on capacitation and acrosomal reaction (Maravilla

- Galvan et al., 2009). Capacitation is a time

- dependent physiological process that is necessary for sperm to undergo befo re they become capable of fertilization. It usually takes place in the uterus and oviduct, but can occur under appropriate culture conditions in vitro . Capacitated sperm can bind the zona pellucida around the oocyte, penetrate it and fuse with (fertilize ) the oocyte. Uncapacitated sperm cannot. Acrosome reaction (AR) consists of an ordered, calcium dependent vesiculation and shedding of the acrosomal vesicle that covers much of the sperm head. It can be visualized using chlortetracycline staining (as w as done in this study). Only acrosome reacted sperm can fuse with the egg membrane. Therefore ARPage 83 of 210 is used as an indicator of capacitation. Progesterone may directly induce acrosome reaction at the sperm membrane level in vitro . Once AR occurs, sperm ten d to slow down and die (run out of energy). Therefore, in a mixed culture there will be continuously changing populations of active capacitating sperm and dying post capacitation/AR sperm. Changes in sperm motion called “hyperactivation” accompany capaci tation and are also necessary for sperm to penetrate the zona pellucida. This study suffers from not reporting hyperactivated motility. The latter would help distinguish between physiologically AR sperm and dead sperm (both of which stain similarly with CTC because they lack an acrosome).

In 2009 Maravilla

- Galvan and coworkers evaluated the impact of atrazine exposure at concentrations of 0 (control), 8, 20, and 40 μM. In general, the percentage of uncapacitated acrosomes ( i.e., with intact acrosomes) and percentage of motile sperm declined in a dose

- dependent manner while the percentage of acrosome reacted sperm increased. There could be two interpretations for this finding. The authors’ conclusion was that atrazine stimulated or accelerated acrosom e reaction in vitro leading to a decrease in motility. Conversely, it could be theorized that atrazine results in decreases in sperm viability and that the dead sperm shed their acrosome after death thus appearing to be acrosome reacted. In the absence o f data on the proportion of hyperactivated sperm, it is impossible to distinguish between these two possibilities. Regardless, these data do not provide any information that correlates the findings to adverse reproductive performance nor does it provide a ny rationale for hypothesizing that atrazine selectively targets sperm. Consequently, the relevance of this study for risk assessment purposes is unclear.

Lastly, a study by Hase et al., examined the effect of atrazine on human sperm motility in vitro an d tested the hypothesis that decreases in sperm motility might be related to decreases in ATP availability needed for motility. Sperm from consenting males was treated with 0, 0.03, 0.1, 0.3, or 1 mM atrazine. Oligomycin (0.05 mM)

– a sperm metabolic poi son which uncouples ATP production in mitochondria

– was used as a positive control. As expected, oligomycin decreased sperm motility by approximately 20%. Atrazine also caused a decrease in sperm motility but only at considerably higher concentrations ( 0.3 and 1 mM) than oligomycin. In support of the authors’ original hypothesis, sperm ATP levels were also decreased at the concentration where progressive motility was impaired. Based on these findings, the authors postulate that the decreases in sperm m otility observed in vitro may be the outcome of decreases in ATP availability needed for sperm flagella to move. They also point out that “the effect of ATZ [atrazine] on sperm motility was seen at > 100 μM, exposure levels that probably do not result fro m normal use of the herbicide. It is therefore uncertain whether the observed phenomena are ecologically relevant.” Thus, though this is an interesting hypothesis and finding, its relevance to risk assessment is questionable.

In summary, three studies evaluating andrology parameters have been published since the 2003 IRED. Using either porcine or human sperm samples, they evaluated atrazine’s potential effect on sperm parameters (motility, viability, capacitation, and acrosomal reaction). Although the in vitro data suggest an effect of atrazine on spermPage 84 of 210 motility and/or viability, there is no evidence of a correlation between these findings and impaired reproductive performance. Furthermore, these studies assume that atrazine per se reaches the testes; an assumption that has not been verified. Finally, the concentrations affecting sperm parameters in these studies are high ( ≥ 100 μM) and, as Hase et al . noted, an exposure level not likely to be reached as a result from use of the herbicide under the cu rrent restrictions. Thus, the data in andrology measures, though interesting, provide no information that would suggest that in vivo exposure to atrazine would result in a sperm

- mediated deficit in reproductive performance.

A.4.1 Albanito L, Lappano R, M adeo A, Chimento A, Prossnitz ER, Cappello AR, Dolce V, Abonante S, Pezzi V, Maggiolini M. (2008). G

protein

- coupled receptor 30 and estrogen receptor

- α are involved in the proliferative effects induced by atrazine in ovarian cancer cells. Environ Health P erspect. 116(12):1648

- 55.

This study used in vitro methods to further evaluate the effects of atrazine in multiple cell lines. The hypothesis for the study is based upon previously reported endocrine disrupting effects of atrazine for androgen

- and estrogen

- mediated processes following in vivo and in vit ro exposures. Using a series of molecular approaches, the study evaluates a number of potential cellular mechanisms to include potential effects on estrogen receptor expression and other estrogen target genes, extracellular signal

related kinase (ERK) pho sphorylation, and cell proliferation.

Studies were conducted using human BG

- 1, MCF

- 7 (breast cancer), Ishikawa (human endometrial), and SkBr3 (breast cancer) cell lines. BG

- 1, MCF

- 7, Ishikawa cells were (transiently) transfected with ER luciferase repo rter plasmids, and SkBr3 was co

transfected with ER **α** expression plasmid and ER

- luc. All transfected cell line were maintained for 24 hrs., and then exposed to solvent, estradiol (100 nM), atrazine (1

– 10 μM)m, ICI ER antagonist (10 μM), or combinations o f ICI + E2, ATR + E2 for an additional 24 hrs. Figure A

- 2 : Treatment Groups shows that atrazine did not stimulate luciferase expression or block E2 induction in any of the cell lines at concentrations up to 10 μM. A study to determine whether or not atrazine regulated ER **α** expression was conducted using BG

- 1 cells, and results demonstrated that atrazine (1 μM) did not alter mRNA or protein concentra tions of the receptor (Fig. 3) as compared to the down

regulation of these measures in those cells exposed to estradiol. In addition, the study confirms that atrazine does not bind directly to the ER as previously reported by Cooper et al. 2007. Together , these data demonstrate that atrazine does not bind or activate ER **α**

. In experiments using the triated

- water release assay, atrazine did not alter aromatase activity or gene expression in the BG

- 1 cells following a 40

- hr exposure to 1 μM. This finding i s in contrast to that reported by Sanderson et al. (2000) for the H295R cell line. However, the current study reports only aromatase activity as % of controls. Without absolute values for aromatase activity of the controls, it is difficult to assess whet her or not the BG

- 1 cells exhibit constitutively high aromatase activity as observed in the R2C Leydig cell line (Heneweer et al., 2004) that would prevent further amplification byPage 85 of 210 exposure to atrazine. In addition, the current study does not provide a po sitive control for either aromatase activity or gene expression.

To test the hypothesis that atrazine may generate rapid signaling via second messenger systems such as Ca2+, cAMP, nitric oxide, and G

- proteins, which in turn can lead to activation of down stream kinase, an experiment was conducted to evaluate the ability of atrazine to produce effects on extracellular signal

- related kinase (ERK) phosphorylation in the BG

- 1 cells. Figure 5A

- B and 6 show that atrazine (1 μM) stimulated ERK phosphorylation wi thin 20 min., albeit a higher concentration and prolonged time was required as compared with effects of estradiol. A similar effect was observed in 2008 ovarian cancer cells. The ER agonist, ICI, prevented the atrazine and E2 mediated effects on ERK phos phorylation (Fig. 6).

Given the results on ERK phosphorylation, the authors further evaluated the ability of atrazine to up

- regulate the expression of c

- fos, an early gene that responds to a variety of extracellular stimuli, including estrogens. An 1

- hr exposure to atrazine (1uM) significantly enhanced c

- fos and cyclin A mRNA levels (200

- 269% increase as compared with control), and longer exposure (24

- hr) resulted with significant elevations in progesterone receptor, pS2k and cyclin A. The authors conclude “Atrazine is able to stimulate the expression of diverse estrogen target genes without an apparent activation of ER **α** . Additional experiments demonstrated that the atrazine dependent up

regulation of c

- fos was abolished after 2

- hr incubation with ER antagonist ICI, the EGFR inhibitor AG, or the ERK inhibitor, PD. Thus, the authors concluded “in ovarian cancer cells, atrazine involves ER **α** and the EGFR/MAPK pathway to trigger c

- fos protein increase.”

The study further evaluated the effects of atra zine on the G

- protein

- coupled receptor 30 (GPR30), a seven

- transmembrane receptor which is structurally unrelated to the nuclear ER, but has recently been shown to mediate rapid actions of estrogens. The authors have also recently shown that c

- fos stimula tion of E2 occurs through GPR30 and requires ER **α** and EGFR

- mediated signaling in cancer cells (Albanito et al 2007, Cancer Res. 67:1859

- 1866). In the current study, both atrazine

- dependent ERK phosphorylation and c

- fos induction were abolished in BG

- 1, 200 8 ovarian, and SkBr3 cells after silencing of GPR30 with small interfering RNA (siRNA), Fig. 10.

Reviewer’s Comments: This study shows no evidence that ATR binds directly or activate ER

- α in cancer cells. It does offer intriguing evidence that atrazine may bind to a membrane receptor that modifies a signaling pathway associated with c

- AMP, ERK, and P13k activation. The concentrations used to indicate this effect are well within, or below the range of concentrations known to modify the steroidogenesis. The fact that the authors identify GPR30 as the target site is also interesting in that although there is clear evidence that this membrane receptor mediates very rapid (non

- genomic) cellular responses, the finding that the ligand typically used in these experiments is estradiol. This manuscript, in combination with the Suzawa and Ingraham and Higley et al., papers argues that atrazine influences signal transduction and provides an alternative to phosphodiesterase as the membrane associated target molecule for the herbicide.Page 86 of 210 A.4.2 Benachour N, Moslemi S, Sipahutar H, Seralini GE. (2007). Cytotoxic effects of aromatase inhibition by xenobiotic endocrine disrupters alone and in combination. Toxicology and Applied Pharmacology. 222:129

- 140.

The purpose of this study was to evaluate the in vitro effects of known endocrine disrupting chemicals on aromatase and cytochrome P450 reductase activities, the later being the second moiety of the aromatase enzyme complex that serves as the electron don or during the conversion of androgens to estrogens. Chemicals (aroclor 1254, atrazine, o,p’

- DDT, vinclozolin, p,p’DDE, bisphenol A, chlordecone, nonylphenol, tributylin oxide, and diethylstilbestrol) were tested in both human placental microsomal and puri fied equine testicular aromatase preparations, as well as in human embryonic 293 cells (transiently) transfected with human aromatase cDNA.

Human embryonic 293 cells (50,000/well) were grown in 24

- well plates to 80% confluency and transfected with human aromatase cDNA 48 hrs prior to exposure to test chemicals. An initial evaluation of cell viability following exposure to each chemical (up to 500 μM) was determined using the MMT method. Following exposure to the test chemicals, media was removed, cells were washed with serum

- free media, and tested for aromatase activity using the tritiated

- water release assay. Specifically, the cells were incubated for 45 min. with 3H

- androstenedione, a substrate for aromatase. 3H

water, released during the conversion of each molecule of androgen to estrogen is a direct measure of estrogen production and enzyme activity. Additional assays were conducted to determine the inhibitory effects of the test chemicals on aromatase activity in human placenta microsomes and equ ine testicular preparations containing the full aromatase (reductase) enzyme complex. Direct effects on the reductase moiety of the aromatase complex was tested by measuring the absorbance corresponding to the reduced form of cytochrome c in the presence of NADPH + . For this assay, reductase (isolated from equine testes) was preincubated with the test chemical for 15 min. prior to initiation of the reaction.

In general, results from the study are difficult to interpret. Initial experiments to detect in hibitory effects on aromatase activity in human placental microsomes were conducted for each test chemical at a single high dose of 500 uM . Atrazine had no inhibitory effect at 500 uM. Chemicals that reduced aromatase activity by 50% were further tested at concentrations ranging from 10

– 1000uM. BPA inhibited reductase activity, albeit the IC50 was exceptionally high, 1250 uM. The authors report “a slight increase in reductase activity in the presence of atrazine” but the actual concentration respons ible for the effect is unclear. Viability of the embryonic cells was affected after 24 hrs in culture at chemical concentrations ≥ 50 uM. Thus, maximal concentrations used for the aromatase assays in the cultures was 20 uM. Binary mixtures were tested for some of the chemicals, but again, data are difficult to interpret. Fig. 7 shows that atrazine significantly inhibited aromatase activity in the embryonic cells.Page 87 of 210 Reviewer’s Comments: In conclusions, the data reported by Benachour et al. are not usefu l for the current assessment of atrazine due to an incomplete documentation of methods, doses, and results. Atrazine is fairly insolubility at concentrations higher than 50 uM, especially in media. Sections of the discussion are poorly written and cite d ata from other studies incorrectly ( e.g. , Sanderson, 2000; Crain 1997).

A.4.3 Fan W, Yanase T, Morinaga H, Gonda S, Okabe T, Nomura M, Hayes T, Takayanagi R, Nawata H. (2007a). Herbicide atrazine activates SF

- 1 by direct affinity and concomitant co

- activa tors recruitments to induce aromatase expression via promoter II. Biochemical and Biophysical Research Communications. 355:1012

- 1018.

The authors were interested in confirming previous findings that atrazine induces aromatase expression via promoter II (ArPII) in a SF

- 1 (steroidogenic factor 1) manner. They were also interested in studying binding of atrazine to the receptor, and whether affinity is independent of the ligand binding domain. The in vitro assays utilized H295R cells (human adrenocortic al cancer cells), HIH

- 3T3 fibroblasts cells, and KGN tumor cell line (human ovarian granulose

- like tumor cell line). A luciferase gene reporter system was examined. Information on the construction of the human CYP19 promoter II luciferase reporter and hu man SF

- 1 reporter was provided.

The in vitro experiments with H295R cells and luciferase gene reporter system showed that the ArPII reporter responded (dose

- dependently) to atrazine (10 - 7 to 10 - 5 M) without exogenous SF

- 1 co

- expression. To confirm that SF

- 1 is essential for atrazine stimulation, endogenous SF

- 1 in H295R cells were knocked down by siRNA. The results indicated a complete loss of responsiveness of ArPII to either atrazine (10 - 5 M) or simazine (10 - 5 M). In the presence of exogenous SF

- 1, atrazine and simazine induced ArPII in the normally non

- responsive KGN cells. The authors also noted that both atrazine and simazine were not cytotoxic and didn’t affect SF

- 1 gene expression.

D ata were presented to demonstrate that protein kinase a (PKA) and liver cell homolog

1 (LRH

- 1) are not essential for atrazine stimulation. To assess if the affinity between SF

- 1 and atrazine is followed by co

- activator recruitment, mammalian two hybrid

- as says for SF1

- TIF2 interaction were performed. Results suggested that atrazine enhanced the interaction between SF

- 1 and its co

- activator TIF2. The authors concluded that atrazine

- SF

- 1 affinity is coupled with enhanced receptor

- co

- activator interactions. To examine the functional domain of SF

- 1 in mediating atrazine stimulation over ArPII, mutations were introduced to the DNA binding domain (DBD) and the ligand binding domain (LBD). Results revealed that mutations in DBD, but not LBD of SF

- 1 alter atraz ine stimulation. The authors concluded that atrazine binds to and activates SF

- 1 to induce ArPII.

Reviewer’s Comments: Fan et al 2007a, b showed that atrazine binds directly to SF

- 1 and induces aromatase expression in vitro . It should be noted, however, that after aromatase activity is not affected after in vivo exposure in mammalian species.Page 88 of 210 A.4.4 Fan W, Yanase T, Morinaga H, Gondo S, Okabe T, Nomura M, Komatsu T, Morohashi K, Hayes TB, Takayanagi R, Nawata H. (2007b). G Atrazine

- induced aromatase expr ession is SF

- 1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans. Environ Health Perspect. 115(5):720

- 7.

This in vitro study focused on the role of steroidogenic factor 1 (SF

- 1) in atrazine

induced aromatase e xpression. A luciferase reporter driven by the SF

- 1 dependent aromatase promoter (ArPII) was used to examine activation of this promoter by atrazine and simazine. Atrazine

- responsive adrenal carcinoma cells (H295R) and atrazine non

responsive cells, ( i.e

. , HIH

- 3T3 fibroblasts cells, and KGN tumor cell line (human ovarian granulose

- like tumor cell line)) were examined. A luciferase gene reporter system was used for the transfection assays.

Atrazine

- responsive H295R cells expressed 54 times more SF

- 1 th an the nonresponsive KGN cells. Exogenous SF

- 1 conferred aromatase responsiveness to atrazine and simazine in KGN cells. The results of the chromatin

- immunoprecipitation (ChIP) assay indicated that atrazine and simazine increased binding of SF

- 1 to ArPII

. Reducing the aromatase promoter II to 516 base pairs did not affect atrazine

- induced aromatase response. However, a mutation in the SF

- 1 binding site on promoter II (AFFTCA to ATTTCA) eliminated the responsiveness to atrazine and simazine treatment.

The authors concluded that atrazine binds directly to SF

- 1, showing that atrazine is a ligand for this “orphan” receptor. The results of this study are supported by another study conducted by these investigators (Fan et al. 2007b).

Reviewer’s Comments : Fan et al 2007a, b showed that atrazine binds directly to SF

- 1 and induces aromatase expression in vitro . It should be noted, however, that after aromatase activity is not affected after in vivo exposure in mammalian species

A.4.5 Heneweer M, van den Berg M, Sanderson JT. (2004). A comparison of human H295R and rat R2C cell lines as in vitro screening tolls for effects on aromatase. Toxicol. Lett. 149(2):183

- 194.

The purpose of this study was to compare two cell lines, rat Leydig cell carcinoma (R2C ) and human adrenocorticocarcinoma (H295R) cells, for their suitability as a screening method to detect effects on aromatase expression and catalytic activity following exposure to selected xenobiotics. For each cell line, aromatase activity using the tri tiated

- water release assay and promoter

- specific mRNA levels were measured following exposure to prochloraz, fadrozole, epoxyconazole, atrazine, 8

- bromo

- cyclic adenosine monophosphate (8

- Br

- cAMP), prostaglandin E2, phorbol 12

- myristate 13

- acetate, and dex amethasone. This study is unique in that it is one of the first efforts to evaluate changes in aromatase activity of the test chemicals as well as the indentify the specific promoter regions of the CYP19 gene that is affected during exposure.Page 89 of 210 For all ex periments, cells were cultured in 24

- well plates, and exposed to each test chemical (1

– 10 uM,) for 24 hr prior to assay for aromatase. These cells were used to evaluate effects on aromatase activity that required mRNA synthesis of the CYP19 gene and/or other transcription factors. In contrast, another group of untreated cells were exposed to the test chemicals only during the 3

– hr incubation period of the aromatase assay to evaluate effects related to direct (rapid) inhibition of the enzyme through co mpetitive or non

- competitive binding of the test chemicals to the enzyme binding sites, binding to allosteric sites of the enzyme causing conformational changes and loss of enzymatic activity, or alteration of other posttranslational events that generally occur rapidly. Appropriate controls were used to demonstrate the specificity of the aromatase assay and measurement of promoter

- specific mRNA levels; solvent controls, 4

- OH

- androstenedione (known inhibitor of catalytic activity of aromatase), and 8

- BR

- cAM P (inducer of aromatase activity). Cell viability was evaluated using the MTT reduction assay.

Results from positive and negative controls confirm assay specificity and demonstrate the precision of the methods to detect significant differences in aromata se activity and gene expression in both cell lines. Atrazine (0.3, 3 and 30 uM) caused a dose

dependent increase in aromatase activity (LOEL 10 uM) in the H295R cells only when exposed for 24 hrs. (Fig.5) indicating that gene transcription and/or other l onger

- term cellular event was required. While an increase in the amplification response of I.3 and pII

- containing aromatase transcripts, the change as shown in Fig. 6 was not significantly different from control or 8

- Br

- cAMP (100 uM). The authors sugges t that the change in these particular promoters may be related to an increase in cAMP, as this transcription factor is crucial for stimulation of this particular promoter. In contrast, no effects on aromatase activity was observed in the R2C Leydig cells following 3 or 24

- hrs of exposure to atrazine or 8

- Br

- cAMP. This cell line, believed to be under the primary regulation of promoter pII, is also simulated by cAMP. The authors report that basal aromatase activity in the R2C cell line is very high as co mpared to that of the H295R cells, and that it is possible that one or more aromatase regions are fully activated, leading to constitutively high aromatase expression in the R2C cells with no further room for induction by chemicals.

Reviewer’s Comments: In conclusion, these data support earlier findings of atrazine

dependent increases in aromatase activity in the H295R cell line, and provide some insight on the possible cellular regulation of aromatase during the 24

- hr exposure period. It is clear that a trazine does not alter enzymatic activity through a rapid (competitive or non

- competitive inhibition, allosteric modification ) alteration of the enzyme. However, failure to evaluate other promoters within the H295R cells is a limitation of this study, an d stops short of providing firm (significant) correlation between increased transcripts with I.3 and pII with elevated aromatase activity. In addition, the study demonstrates that the R2C Leydig cell line is not suitable for screening chemicals for identi fication of induction of aromatase gene expression.Page 90 of 210 Aromatase activity (AA)

LOEL 10 uM, NOEL 0.3 uM) : H295R cells displayed dose dependant increase in AA (0.3 , 10 and 30 uM) with 3

- fold induction as compared to control at highest dose.

R2C cells = no sign. effect on AA or cell viability (at 30uM), NOEL 30 uM (Authors suggest lack of effect was likely due to constitutively high aromatase expression as compared with that observed for the H295R cells.

Promoter

- specific mRNA Levels H295R cell s

– Following 24 hrs exposure, an increase in I.3 and pII transcripts was observed in cells treated with 30 uM, but was not sign. different than control or 8

- Br

cAMP. (Thus, it is still unclear whether or not cAMP and/or activation of these promoters are totally responsible for elevated aromatase activity). :

A.4.6 Higley EB, Newsted JL, Zhang X, Giesy JP, Hecker M. (2010). Assessment of chemicals effects on aromatase activity using the H295R cell line. Environ Sci Pollut Res Int. Jan 20. (Epub ahead of print).

This study compared the effects of seven model chemicals (atrazine, forskolin, prometon, ketoconazole, aminoglutethimide, letrozole, and prochloraz) on aromatase activity and steroid production (estrogen, testosterone) in H295R human adenocarcino ma cells. The suite of chemicals selected for the study were selected because they were chemicals known to induce aromatase activity through up

- regulation of gene expression and/or transcription factors. Using a tritiated

- water release aromatase assay to quantify the conversion of radiolabeled androstenedione to estrone, the authors tested an approach to determine whether or not a given chemical affected aromatase directly (e.g., immediate competitive inhibition of enzyme), indirectly (e.g., altered activi ty requiring a longer exposure time), or a combination of both direct and indirect effects. Measurement of estrogen and testosterone allowed an evaluation of the predictability of these endpoints for effects on aromatase activity in the cultured cells.

F or each experiment, H295R cells were cultured in 24

- well plates, 300,000 cell/ml, and allowed to attach for 24 hrs prior to treatment. Media was changed and cells were exposed to test chemicals for an additional 48 hrs. DMSO ( ≤ 0.1% v/v) was used as solv ent, and six concentrations of the each chemical (0.01 to 10 uM) along with solvent controls were tested in duplicate or triplicate. MMT assay used for detection of cytotoxicity. At the end of each experiment, culture media was collected for hormone

assa y (ELISA), and the cells were further subjected to the tritiated

- water release aromatase assay for 1.5 hrs. To differentiate between direct, indirect or a combination of direct and indirect effects on aromatase activity, the exposure of the cells to the t est chemical was varied in one of 3 ways: (1) indirect effect

- no further exposure of the cells during the 1.5 hr. incubation, (2) combination of direct and indirect effects

- cells exposed for 48 hrs in culture and then again during the 1.5 hr assay, and (3) untreated cells during the 48 hrs culture were exposed to the test chemical only during the 1.5 hr assay period.Page 91 of 210 Reviewer’s Comments: Overall, the results of this study indicate that the increase in estradiol observed in the H295R cell assay was not due to a direct increase in aromatase activity. Atrazine, like forskolin (which activates the enzyme adenylyl cyclase and increases the intracellular levels of cAMP) enhanced steroidogenesis as evidence by increased T and E. In contrast, when atrazine an d forskilin were tested in the “direct” tritiated water assay, there was no apparent change in aromatase activity (even at concentrations greater than the concentrations that changed estradiol production. These observations are important because they demo nstrate that atrazine does not interact directly with the enzyme aromatase (as indicated earlier by the paper of Sanderson using the same cell line) and casts further doubt on the hypotheses that any adverse effects of the chemical are mediated through act ivation of aromatase.

In addition, significant elevations of (indirect) aromatase activity were only observed following a longer exposure for both of these chemicals. The authors conclude, “The hormone and aromatase activity profiles for forskolin and at razine were comparable, and suggest similar mechanisms of activity even though exposure to forskolin resulted in greater concentrations of estrogen and testosterone, and greater aromatase activity at lower concentrations than observed for atrazine.” In co ntrast, known inhibitors of aromatase (prochloraz, letrozole, aminoglutethimide, ketoconazole) caused decreases in estrogen, testosterone and (direct) aromatase activity measured following the 1.5 hrs exposure. The novel finding of increased (indirect) ar omatase activity following longer exposures to these inhibitors suggests that a compensatory feedback regulation of aromatase gene expression may also occur in response to the low estrogen concentrations. Thus, the authors conclude that multiple measures of aromatase activity to evaluate immediate (direct) effects on the enzyme as well as longer term (indirect) effects should be measured in using in vitro culture systems such as the H295R cell line.Page 92 of 210 Figure A

- 1 : Effect of Atrazine Exposure on Aromatase Activity and Hormone Levels (A1, A2) Atrazine: 48

- hrs. exposure, 0.01

– 100 uM, E2, 7.2X increase (100uM), LOEL (1 uM), NOEL (0.1 uM) T, 1.4X increase (100 uM), LOEL (10 uM), NOEL (1.0 uM) Direct AA: no effect (100uM) Indirect AA: increase LOEL (1 uM), NOEL (0.1 uM)

Limitations of study:

Data for all endpoints reported as fold

- change, with no actual values for means **±** SEM for any observations for steroid concentrations or aromatase activity. Without control data , it is difficult to gauge how consistent data are among experiments within the study or compare with data reported elsewhere.
The large discrepancy between the fold changes in AA and E2 (ATR (100 uM) AA was 2.25X versus 7.2X for E2 as compared to respect ive controls) in Fig 17. is believed to be a reflection of different collection times, e.g , AA is that change which occurs in tritiated water release over a 1.5 hr period, versus E2 production over the 48 hrs in culture.
Although positive controls used t o demonstrate specificity of aromatase assay (e.g., radio inert androstenedione and fadrozole), no concentrations for androstenedione reported and vague information on that used for fadrozole.
Solubility of ATR (100uM) is questionable.Page 93 of 210 A.4.7 Holloway, A. Anger, D., Crankshaw, D., Wu, M, and Foster, W. 2008. Atrazine

- induced Changes in Aromatase Activity in Estrogen Sensitive Target Tissues. Journal of Applied Toxicology. 28:260

- 270.

The purpose of this study was to determine the effects of atrazine tr eatment on aromatase activity in cells from estrogen sensitive target tissues in the ovary and endometrium.

The effect of atrazine on aromatase activity was measured in human granulosa

- lutein cell cultures, cells that abundantly express aromatase, and end ometrial stromal cell (ESC) cultures, cells that do not express aromatase. Aromatase activity was quantified by the tritiated water method and the specificity of the assay was confirmed by co

incubation with 4

- hydroxyandrostenedione, an irreversible inhib itor of the catalytic activity of aromatase, to block the formation of tritiated water. The cells were treated for 24 hours with increasing concentrations (1 nM to 1μM) of atrazine. Prostaglandin E 2 (PGE 2 , 1 μM) and vomitoxin (a selective cyclooxygenase

- I I inducer, 250 mg/ml) were used as positive controls.

In the granulosa

- lutein cell cultures, atrazine treatment induced a dose

- dependent increase in aromatase activity (p=0.007). Aromatase activity in atrazine treated (1 nM

0.1 mM) granulosa

- lutein cel ls (GC) was increased more than 2

- fold compared with control cultures, with significant aromatase activities at 0.1 and 1.0 μM. Atrazine treatment had no effect on ESC aromatase activity at any concentration tested. Atrazine treatment had no effect on GC or ESC protein concentrations, which indicated no change in cell number, suggesting that the atrazine induced changes in aromatase activity were not due to cell proliferation. The positive controls PGE 2 (1.0 μM) and vomitoxin (250 ng/ml) induced a signi ficant increase in aromatase activity (132 ± 10 and 149± 13% of control, respectively, p<0.05). There was also no effect of atrazine treatment on human recombinant aromatase activity in cell free test system. It was concluded that μM concentrations of atra zine can increase aromatase activity of human granulosa cells but not ESC and this effect is not elicited at the enzyme level.

Reviewer’s Comments: The authors concluded that atrazine treatment induced an increase in aromatase activity in human luteinized granulosa cells, however, the concentrations needed to induce the observed change is considered to be outside the range likely to be achieved in human tissues and, therefore, is unlikely to pose a health threat through this mechanism.

A.4.8 Laville N, Balaguer P, Brion F, Hinfray N, Casellas C, Porcher JM, Ait

Aissa S. (2006). Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG

- 3 cell line. Toxicology. 228:98

- 108.

This study evaluates 30 pesticides for their ability to modulate aromatase activity in the human choriocarcinoma JEG

- 3 cell line. Pesticides were selected based upon chemical structure and environmental relevance, but also included a number of known aromatasePage 94 of 210 inhibitors and inducers. Atr azine was among one of the reference pesticides used in this study. By using the tritiated water release assay after both short (2 hr) and long (24hr) exposures to each test chemical, the authors were able to identify either inhibition or induction (requ iring longer term exposure) of aromatase enzymatic activity. Pesticides that were newly found to induce aromatase activity were further evaluated for their action at the transcriptional level with measurement of CYP19 mRNA and through the retinoic acid re ceptor signaling pathway.

JEG

- 3 cells were seeded in 24

- well plates, density of 50,000 cells/well, and allowed to attach for 24 hrs. before changing media and exposing the cells to the test chemicals (1, 3, 10 uM) for an additional 24 hrs. Media was coll ected at the end of the exposure period, cells were washed once with media, and further elevated for aromatase activity. The tritiated water release assay was used to quantify aromatase activity in cells exposed to test chemicals for 24 hrs (to detect po ssible effects of chemicals on aromatase gene expression, or changes at transcription or (post) transcription levels), and in untreated cells exposed to the test chemical for only 2 hrs (e.g., direct competitive effects on the aromatase enzymatic complex). OH

- Androstenedione, an irreversible inhibitor of aromatase activity was used to demonstrate time

- and concentration

- dependent inhibition. Forskolin and TTNPB were used as positive controls for up

- regulation of aromatase expression. Cell viability was d etermined using the methyl

- thiazol

- tetrazolium (MMT) assay.

Reviewer’s Comments: While neither atrazine nor forskolin had any effect on aromatase activity following a 2 hr exposure, both of these chemicals increased aromatase activity after 24 hrs of exp osure. Approximately a 2

- fold increase in aromatase activity was observed in cells treated with 10 uM atrazine, but no change in CYP19 mRNA was observed.

Atrazine: JEG3 cells, doses 1, 3, and 10 uM (24 hrs) LOEL: 10 uM NOEL: 3 uM

Strengths of study

in vitro studies demonstrating that atrazine does not alter aromatase enzyme competitive inhibition, but ra ther requires longer exposure time such that enzyme stability, synthesis and/or post

- transcriptional events can be altered. : Use of positive controls to demonstrate specificity of aromatase assay and CYP19 mRNA measurements. This study supports other

A.4.9 Rayner, J., Wood, C., and Fenton, S. (2004) Exposure Parameters Necessary for Delayed Puberty and Mammary Gland Development in Long

- Evans Rats Exposed in Utero to Atrazine. Toxicology and Applied Pharmacology 195:23

- 34.

This study looked at the effects on delayed puberty and mammary gland development in rats exposed in utero to atrazine. 14

14 This review will concentrate on the assessment of delayed puberty onset. The discussion of the mammary gland development will be deferred until the September SAP meeting. Groups of 20 timed

- pregnant Long

- Evan s ratsPage 95 of 210 were gavaged on gestational days (GD) 15

- 19 with atrazine (purity 97.1%) at doses of 100 mg/kg bw or vehicle control (C). On PND 1, half of each litter was cross

- fostered, creating four treatment groups: C

- C, ATR

- C, C

- ATR, and ATR

- ATR (dam

- milk sou rce, respectively). Litters were weighed and equalized to 10 pups on PND4. On PND 22 (weaning), animals were weighed and separated into 2

- 4 females/cage. Some females were sacrificed on PND 4, 22, 33, 40 and 58. The data from a treated

- dam’s offspring

wer e evaluated with the dam (two pups/dam or litter) as the experimental unit, with the exception of vaginal smears.

On PND 29 females (n>29/group) were evaluated for vaginal opening (VO). The PND of complete VO and body weight on that day were recorded. D aily vaginal smears were collected beginning the day after VO for each animal until PND 58 to determine cyclicity patterns. Female pups (n=14

- 16) were sacrificed on PND58. Blood was collected and the pituitary gland, ovaries, and uterus were weighed and pr epared for histopathology. Serum luteinizing hormone (LH), prolactin (PRL), and thyroid stimulating hormone (TSH) were analyzed.

Pups in the ATR

- ATR group were statistically significantly smaller than those in the control group on PND4 (p<0.01) (8.2±0.1 g ATR

- ATR vs 8.7±0.1g, C

- C), however, their mean weight was reduced 5.7% from control animals, which was within normal range. There was no difference in body weight among groups at PND 22, 33, 40 or 58. A significant delay (p<0.02) in VO was seen only in a nimals nursing from ATR

- treated dams (C

- ATR 35.4±0.6 days, and ATR

- ATR 37.1±0.5 days vs C

- C 33±0.4 days). Mean body weight on day of VO was significantly (p<0.002) increased in the ATR

- ATR group (~10%, estimated from graph), but not in the C

- ATR group, co mpared to C

- C group. There were no significant differences in the number of consecutive normal estrous cycles between VO and PND 58 or in the number of animals demonstrating persistent estrous (PE) or persistent diestrous (PD). However, while not statisti cally significant, there appeared to be a lower number of animals with irregular cycles in the ATR

- ATR group (3%), compared to controls (23%) and the number of animals with normal cycles (2

- 5 days) appears to be increased in the ATR

- ATR animals, compared t o controls.

No significant treatment

- related changes were seen in circulating hormones (serum LH, PRL, and TSH) in any treatment group at PND 33, 40 or 58.

At PND 58, all atrazine exposure groups had increased pituitary glands; however only the pituitar y glands of the animals in the ATR

- ATR group were significantly increased, compared to controls. There were no statistically significant differences in reproductive organ weights among the groups.

Reviewer’s Comments: The researchers in this article demonstrate that atrazine in utero and perinatal exposure to atrazine can lead to a delay in vaginal opening in female rats. These findings are consistent with those reported by Laws et al. in 2000

an d 2003. It should be noted, however, that these effects are seen at substantially higher dose levels than those used as points of departure in the Agency’s human health risk assessment.Page 96 of 210 A.4.10 Suzawa, M and Ingraham, HA. 2008. The herbicide atrazine

a ctivates endocrine gene networks via non

- steroidal NR5A nuclear receptors in fish and mammalian cells. PLoS ONE 3(5): e2117. doi:10.1371/journal.pone.0002117.

* In this evaluation, only the results pertaining to mammalian cells are discussed. The purpo se of this in vitro study was to examine the potential effects of atrazine on NR5A receptors (steroidogenic factor 1 or SF

- 1). The specific interest was on whether Cyp19A1 (aromatase), a known target of NR5A receptors would be selectively activated by at razine. The focus on SF

- 1 was a result of the two papers by Fan et al., who argued that this NR5A receptor was the target molecule for atrazine evoking changes in aromatase activity and potentially the molecular mechanism for many assumed adverse outcom es following exposure to this herbicide in vivo. Cellular reporter assays, utilizing JEG3 human placental cells, were performed. JEG3 cells contain modest amounts of SF

- 1. In one experiment, the effects of in vitro atrazine on luciferase activity after t ransfection of human placental JEG3 cells with aromatase

- luciferase reporter and the parent reporter (pGL3

- luciferase) with mSF

- 1 (mouse SF

- 1) and hLRH

- 1 (human liver receptor homolog) expression vectors were examined. The data revealed a dose

dependent increase in luciferase activity which the authors interpreted as activation of aromatase promoters. The effects were significant at atrazine concentrations of ≥ 1 μM. No activation was reported for the parent reporter or with mutant SF

- 1 binding sites. Atrazine also activated mSF

- 1 in HepG2 (human liver) cells. The authors also provided data demonstrating activation of other SF

- 1 reporters, such as MIS

- Luc, StAR

Luc, and HSD3B

- Luc. The authors concluded that atrazine effects depend on DNA binding and receptor occupancy of the promoter.

In a knock

- down experiment, the authors provided results showing knocking

- down hLRH

- 1 in human HepG2 liver cells attenuated atrazine

- induced activation of the aromatase reporter, which the authors interpreted as demons trating a dependence on this NR5A receptor. In addition, they conclude that all NR5A subfamily receptors will be responsive to atrazine.

Atrazine treatment was without effects on the estrogen responsive zcyp19a2 (ERE

- Luc reporter). No significant acti vation of NR5A was observed with other estrogenic compounds tested.

Several additional experiments were performed to determine the underlying cellular mechanisms of activation of NR5A receptors. These included mitogen

- activated protein protein kinase ( MAPK), phosphatidyl 3

- kinase (Pl3K), G proteins, phospholipase C (PLC) in JEG3 cells. Pharmacological inhibitors of these systems were examined. The results indicated that inhibitors of both the MAPK and Pl3K signaling decreased activation by atrazine, w hereas the inhibitors did not affect G protein or PLC signaling. The finding of phosphorylation of AKT/protein kinase B provided further support for effects on MAPK and Pl3K signaling. Atrazine ( ≥ 1 μM) significantly increased cAMP levels in JEG3 cells.Page 97 of 210 The authors also assessed whether or not atrazine binds directly to SF

- 1 to either increase DNA binding or act as a ligand. Atrazine had no effect on DNA binding activity to a high affinity SF

- 1 binding site (the mMIS binding site was used). Atrazine a lso did not activate a Gal4

- SF

- 1 ligand binding domain fusion construct. The authors concluded that these results suggest that atrazine may not directly interact with NR5A receptors, but instead activates three signaling pathways known to activate NR5A

re ceptors, including phosphorylation of SF

- 1, generation of SF

- 1 ligands, and increased production of cAMP. The authors also reported that atrazine can increase a cluster of genes involved in endocrine signaling in specific cell types; however, very limited data were presented to corroborate these findings.

Reviewer’s Comments: Overall, this study provides some preliminary research on the underlying cellular mechanism of aromatase expression following atrazine in vitro treatment. The study has limitations including lack of details on methodology of luciferase activity and transfection of cells. Only a few poor quality results of PCR analyses and western blots were provided to corroborate the findings. The authors made several references to “data not sho wn” to support their conclusions. The activation of important second messenger pathways, and lack of evidence for direct binding to SF

- 1 does not support the reports of Fan et al ., who conclude that atrazine has a direct SF

- 1 mediated activation of aroma tase and subsequently increase estradiol production. The results indicating an indirect activation of aromatase are also in agreement with the observations of Higley et al reviewed above. The experiment examining the effect of atrazine on zebra fish was p reviously reviewed by the Agency and numerous deficiencies were identified.

A.4.11 Yi D K, Kim P J, et al. 2010. Dose

- and time

- dependent effect of atrazine or its metabolites on aromatase mRNA in vitro. Only a preprint of article available.

The p urpose of this study (available only as a preprint) was to examine the potential effects of dose and duration of exposure to atrazine and its metabolites (diaminochlorotriazine, DACT; deethyl atrazine, DEA; deisopropyl atrazine, DIA) in vitro on the expres sion of aromatase mRNA in two steroidogenic lines, i.e. , human adrenocortical cancer cells (H295R), and human placental carcinoma cells (JEG3). There were no details in the methods section of the article pertaining to the concentrations used and the durat ion of treatment. Based on information in the abstract and figures, atrazine and metabolites (all were reported to be 97% purity and dissolved in DMSO) were administered in vitro at equimolar concentrations of 0, 100 nM, 1 μM or 10 μM for 15 minutes to 72 hours. There were descriptions of cell cultures, RNA extraction and cDNA synthesis, primer design, reverse transcription PCR, and statistical analyses.

Exposure to 10 μM atrazine increased aromatase mRNA expression in H295R cells approximately 2.2 fold after 2 hours and approximately 3

- fold after 4 to 24 hours. Aromatase mRNA expression was increased about 2.5 fold at 48 hours and 2.2 foldPage 98 of 210 after 72 hours. At 1 μM atrazine, there was about a 2

- fold increase in aromatase mRNA expression at 24 hours but not at other time points. No expression was observed at the low concentration of atrazine.

Exposure of H295R cells to 10 μM DEA increased aromatase mRNA approximately 1.5

- fold after 2 to 24 hour time period. Exposure of H295R cells to 10 μM DIA incre ased aromatase mRNA approximately 1.8

- fold after 2 to 48 hour time period. There were no apparent treatment

- related effects with hydroxyatrazine and DACT.

Reviewer’s Comments: The authors concluded that exposure to 10 μM atrazine in vitro resulted i n an increased expression of aromatase mRNA. This in vitro response is dose

- and time

- dependent with peak expression occurring after 4 to 24 hours of exposure. Atrazine metabolites, DACT and hydroxyatrazine, failed to show a consistent response.

A.4.12 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (2010?) Ex vivo effects of atrazines or metabolites on phosphodiesterase activity.

This study concentrated on phosphodiesterase (PDE) activity and whether atrazine and its metabolites inhibit this process, while also noting effects on aromatase in vivo . It provides several theories on the possible MOA regarding the incidence of mammary tumors, delayed onset of puberty, and immune suppression.

The premise that there is an alternative MOA for m ammary tumors in female Sprague

Dawley rats is that mRNA expression and aromatase activity was increased in H295R adrenocortical carcinoma cells when exposed to a minimum of 10μM atrazine. Increased cyclic AMP (cAMP) levels were correlated with the induct ion of aromatase activity by atrazine; increases in cAMP may be mediated either via increases in adenylyl cyclase which catalyzes the conversion of ATP to cAMP, or via the inhibition of PDE which catalyzes the breakdown of cAMP to AMP. Therefore, high con centrations of atrazine in vitro may be mediated by increases in intracellular cAMP, activating adenylyl cyclase, or by inhibiting PDE.

Atrazine, deethyl atrazine (DEA), deisopropyl atrazine (DIA), diaminochlorotriazine (DACT), ammeline (AM), and hydrox yatrazine (hydroxyl

- ATZ) used were approximately 97% pure. IBMX, 3

- isobutyl

- 1

- methylxanthine was used as the positive control. The IMAP phosphodiesterase assay kit was used along with bovine PDE at varying concentrations (0.000025 to 0.5 PDE U/ml in 96

- w ell plates). To determine the effects of addition of atrazine after completion of the PDE reaction, an hour long incubation period was allowed and then to assess the effects of atrazine and its metabolites on PDE activity, the potential inhibitors were ad ded at concentrations ranging from 5pM to 5 nM in 10 μl of assay buffer enzyme with additional 90 minute substrate/incubation period. Competitive inhibition studies were similarly conducted with the addition of varying concentrations of DACT with atrazine . Determining IC 50 values was done using a 4

- parameter log curve and creating sigmoidal standard curves.Page 99 of 210 The results from the optimized assay for PDE inhibition showed a concentration

dependent increase in the relative fluorescence output of the assay at both 30 minutes and 2 hours after the addition of the binding reagent. Maximal output, of 0.025 PDE U/ml was used for all subsequent assays. Addition of atrazine after completion of the enzymatic reaction didn’t have any effect on the fluorescence out put of the assay. Regarding the effects of atrazine and to a much lesser extent its metabolites inhibited PDE in a cell

- free system. The IC 50 for atrazine (39.52 μM) while for IBMX it was 1.749 μM. The IC 50 for DIA was 1371 μM and DEA at 7156 μM were 35 and 181

- fold less potent inhibitors of PDE than was atrazine; all other metabolites were inactive. DACT, however dose

- dependently caused a rightward shift in the dose

- response curve of the atrazine mediated inhibition of PDE activity suggesting to the au thors that although DACT does not inhibit PDE out right, it perhaps modifies the effect of atrazine on PDE. Also at both 5 and 50 μM concentrations of DACT, the IC 50 of atrazine was several folds higher, suggesting competitive inhibitory effects of DACT.

The authors concluded that atrazine at all concentrations which exceed its aqueous solubility limits, is a weak inhibitor of PDE and that its metabolites have extremely weak, or no effect on PDE activity. The primary metabolite of atrazine, DACT was show n to act as a competitive inhibitor of atrazine

- induced inhibition of PDE activity. Although, not supported, the authors elude to the possibility that the inhibition by atrazine of PDE in ex vivo condition, a cell

- free system with a complex mixture of dif ferent PDE isozymes and substrate, could be due to a non

- specific effect of the precipitate on the kinetics of the reaction. The IC 50 for atrazine

- mediated inhibition of PDE activity in this study (40 μM) was vastly different, 22

- fold higher, form the con centration reported by Roberge et al (2004). No true rationale was given for this discrepancy

– however, they went on to describe the many similarities in results from the two laboratories. Even at high concentrations of atrazine there have been no shown increases in aromatase mRNA or enzyme activity in vivo with the exception of a study on juvenile zebrafish. Therefore the study concludes that a general or local increase in the expression of this enzyme in vivo , would not account for the observed effec ts of high doses of atrazine on estrous cycles, mammary tumors or the age of onset of puberty.

Reviewer’s Comments: In mammals, atrazine is NOT acutely toxic (oral LD= 3090 mg/kg) and is only moderately toxic following repeat dosing to rats, mice, rabbi ts, or dogs. Atrazine

- mediated inhibition of PDE ex vivo is unlikely to account for the tissue, sex, strain and species

- specific effects of chronic high

- dose exposure to atrazine. The bottom

- line is that it is unlikely that an atrazine

- mediated inhibitio n PDE activity would occur at environmentally relevant concentrations. The potential human exposure to atrazine is low and equally unlikely to result in PDE inhibition in humans. The study goes on to endorse the NOEL (1.8 mg/kg/day) which is currently us ed to regulate chronic exposure to atrazine and its chlorometabolites and appears to be protective of any potential effects in humans.

A.4.13 Pogrmic

- Majkic K, Fa S., Dakic V, Kaisarevic S., Kovacevic R. 2010. Upregulation of Peripubertal Leydig Cell Ste roidogenesis FollowingPage 100 of 210 24 h In Vitro and In Vico Exposure to Atrazine. Toxicol. Sci. 118(1): 52

- 60

In this study the authors investigated the impact of direct exposure of Leydig cells to atrazine on steroidogenesis by studying changes in the levels of cAM P, androgen, and progesterone synthesis by Leydig cells. They pursued this line of investigation using both in vitro and ex

- vivo approaches. For the in vitro studies, Leydig cells obtained from peripubertal (PND 51) male Wistar rats were exposed to atr azine (98% purity) at concentrations of 0, 0.001, 1, 10, 20, and 50 μM for 24 hours. At the end of the incubation period the culture medium was collected and cells were stimulated with different concentrations of human chorion gonadotropin hormone (hCG), progesterone

or ∆ 4

- androstenedione for 2 hrs. For the ex

- vivo studies male Wistar rats were exposed to 200 mg/kg/day atrazine ( via gavage) for 6 days (PND45

- 50), 3 days (PND48

- 50) and 1 day (PND50). Twenty four hours after the last exposure the animals were sacrificed and Leydig cells collected to measure levels of cAMP and testosterone after the cells were incubated for 2 hrs with hCG.

In vitro studies:

In addition to the assessments on the role of cAMP and PKA in androgen biosynthesis, an evaluation of the mRNA transcripts for genes involved in androgenesis includ ing Luteinizing Hormone Receptor (LHR), Steroidogenic acute regulatory protein (StAR, involved in cholesterol transport), Steroidogenic factor

- 1 (SF

- 1), CYP17A1, 17β

hydroxysteroid dehydrogenase (17β

- HSD), scavenger receptor B

- 1 (SR

- B1), and translocator protein (TSPO) was conducted to ascertain what genes

– if any

– were activated after exposure to 1 or 20 μM atrazine. While SF

- 1 was statistically significantly (p<0.05) increased after exposure to either of the atrazine concentrations ( ↑141 and 191%, r espectively), StAR, CYP17A1 and 17βHSD were only increased at A 24 hr exposure to either 1 or 20 μM atrazine resulted in an increase in basal cAMP levels of Leydi g cells. Interestingly, in hCG

- stimulated Leydig cells the increase in cAMP was only observed after exposure to 20 μM atrazine. Since cAMP has been implicated in the regulation of Leydig cell steroidogenesis, the impact of atrazine exposure on basal prog esterone and androgen production was also evaluated. Increases in androgen production ( ↑31

- 98%) measured as testosterone + dihydrotestosterone levels were statistically significant and dose

- dependent at concentrations ≥ 1μM. In contrast, progesterone lev els exhibited an inverted dose

response pattern with increases in progesterone levels after exposure to 0.001 or 1 μM atrazine ( ↑24 and 41%, respectively) and decreases at concentrations of 20 or 50 μM atrazine ( ↓13 and 22%, respectively). Together these findings lend credence to the hypothesis that androgen synthesis is dependent on cAMP

- protein kinase A (PKA). To further test this hypothesis, hCG

- stimulated or unstimulated Leydig cells exposed to 0 or 20 μM atrazine for 24 hrs were subsequently treated with PKA

- inhibitor H89 (10 μM). While the atrazine induced increase in cAMP was not affected, androgen production was markedly reduced ( ≈60%) after H89 treatment. Thus, under the conditions of this study, the pathway leading to androgenesis is blocked at the PKA step suggesting this kinase plays a critical role in in vitro androgen synthesis.Page 101 of 210 the 20 μM concentration ( ↑162, 247, and 153%, respectively). In contrast, neither LHR, SR

- B1, or TSPO were affected by atrazine exposure (data not shown). Given that 17β

HSD and CYP17A1 trans cript levels were increased after exposure to 20 μM atrazine, the investigators evaluated whether this translates into an increase in the protein levels of these enzymes by measuring the conversion of progesterone to testosterone as well as the conversion of ∆ 4

- androstenedione to testosterone. After a 24 hours exposure to 0, 0.001, 1, 20 or 50 μM atrazine, Leydig cells were treated with either progesterone or ∆ 4

- androstenedione at concentrations of 0.25, 0.5, or 10 μM for 2 hrs. Exposure to atrazine at ≥ 20 μM atrazine resulted in an increased conversion of progesterone to testosterone ( ≈227%, p<0.05). For cells receiving 0.25 μM ∆ 4

- androstenedione, atrazine exposure at concentrations ≥ 1 μM resulted in a statistically significant increase in testosteron e although the response is not as robust as for the progesterone to testosterone conversion. It is noteworthy that for cells receiving 0.5 or 10 μM of the steroid substrate ( ∆ 4

– androstenedione) an increase in testosterone levels was only noted at the 20 μM atrazine level.

In the ex vivo experiments, peripubertal male Wistar rats were exposed by gavage to 0 or 200 mg/kg/day atrazine for 1(PND 50), 3 (PND48

- 50), or 6 days (PND 45

- 50). Twenty four hours after the last exposure Leydig cel ls were isolated and cAMP and testosterone levels measured. After a single exposure, cAMP and testosterone levels were increased ( ↑40%) regardless of whether the cells were hCG

- stimulated or unstimulated. Peak increases in both cAMP and testosterone were reached after 3 days of atrazine exposure in unstimulated cells ( ≈↑150

- 180%) and hCG

- stimulated cells ( ≈↑100%). After 6 days, however, cAMP levels were comparable between the control and atrazine exposed groups. In the case of testosterone, the levels in the atrazine treated group continued to be elevated relative to control but decreased to the levels observed after a single exposure. Ex vivo experiments

Reviewer comments : In general, this appears to be a well

- conducted study. However, details on the study design and resul ts are lacking. As an example, the number of rats treated during the ex vivo experiments is not provided and findings at the 50 μM atrazine concentration in the in vitro experiments are only reported for the progesterone or androstenedione

- stimulated test osterone synthesis and the basal androgen and progesterone synthesis assessments. Given that a bidirectional dose

- response was observed during the basal progesterone synthesis assessment, inclusion of the entire dose

- response curve for these experiments i s considered to be crucial. In conclusion, this set of experiments suggests that cAMP signaling and subsequent PKA activation are critical in androgenesis by catalyzing the upregulation of genes involved in steroidogenesis such as SF

- 1, StAR, CYP17A1, and 17βHSD. Regardless, the ex vivo data effects reported in this study occurred at doses that are approximately 2 orders of magnitude higher than the point of departure currently being used for regulatory purposes. Thus, the utility of these data for risk assessment purposes is limited.

A.5 NEUROTOXICITYPage 102 of 210 Summary: Neurotoxicity

The preponderance of the data available to date indicates that atrazine exerts many of its toxicologically relevant effects through perturbations of the neuroendocrine system. Many of the reproductive effects seen after atrazine exposure appear to be the sequelae of perturbations to the HPG and/or HPA axes. Consequently, there is significant interest in the potential effects of atrazine exposure on the nervous system per se.
Over the last seven years, several studies have been conducted evaluating pote ntial atrazine

- induced effects on the dopaminergic and somatostatinergic systems as well as GnRH releasing neurons. Studies on the dopaminergic system by Coban and Filipov (2007) and Rodriguez et al. (2005) corroborate previous findings on the effect of at razine on the dopaminergic system. The work by Rodriguez et al. showed that 9

- month old male rats exposed for 6 months to 10 mg/kg/day atrazine in the diet exhibited increased locomotor activity, decreased striatal dopamine, reduced norepinephrine in the prefrontal cortex, decreased hypothalamic serotonin, and loss of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH

- ) cells in the ventral tegmental area and the substantia nigra pars compacta. It should be noted that the changes in neurotransmitter levels were slight. In 2007, Coban and Filipov studied the effect of atrazine on male mice after a 14 day exposure to dose levels ≥ 5 mg/kg/day. The researchers reported a statistically significant reduction in the number of TH+

neurons in the ventral tegmental area (day 64 of the experiment) as well as a decrease in the number of TH + neurons in the substantia nigra pars compacta was reported at 25 mg/kg/day at days 22 and 64. Noticeably lacking was a dose

- response for either one of the se findings. Coban and Filipov (2007) also concluded that there was a dose

dependent decrease in the levels of dopamine and its metabolites in the striatum. Based on these findings, both research groups suggested that exposure to atrazine may be related to the etiology of Parkinson’s disease. Both of these studies have limitations that confound the interpretation of the findings. These limitations are fully described in the study summaries found in Appendix A. The implication that these data suggest a l ink between atrazine and Parkinson’s disease is not supported by the data given the non

- specificity of the atrazine

- induced neuronal loss and the lack of histological and behavioral features characteristic of Parkinson’s Disease.

Parkinson’s disease is a neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra. The data reported by Rodriguez et al. (2005) shows loss of non

- dopaminergic neurons and dopaminergic neurons following atrazine treatment in the rat. The subpopulation of non

- dopaminergic neurons includes GABA

- ergic, calbindin, and cholecystokinin

- like immunoreactive neurons. Other potential cell types may include glial cells. The data presented by Coban and Filipov (2007) reveal a statistically significant loss of dopaminergic neurons in the ventral tegmental area in the mouse at 5 mg/kg/day on day 64 of the experiment, while the loss in the substantia nigra pars compacta was significant at 25 mg/kg/day but not at 5 mg/kg/day. The loss of dopam inergic neurons at the high dose at day 64 was comparable between the two brain regions. The ventral tegmental area containsPage 103 of 210 dopaminergic cell bodies that comprise the mesocorticolimbic system that is involved with functions such as cognition. The primary and major loss of dopaminergic neurons in Parkinson’s disease involves the substantia nigra. The nonspecific neurotoxicity of atrazine contrasts with selective striatal dopaminergic neurotoxins such as 6

- hydroxydopamine and MPTP.

In addition to the non

- specificity of the atrazine

- induced neuronal loss, another characteristic not seen after atrazine exposure is the suite of neurobehavioral and histological features associated with Parkinsons’s disease. Histologically, Parkinson’s disease is characterize d by the presence of intracytoplasmic inclusions, Lewy bodies, formed mainly by α

- synuclein and ubiquitin. None of the available studies have demonstrated any aggregation of proteins that form Lewy bodies after atrazine treatment. Behavioral abnormalitie s noted in Parkinson’s disease include tremor, bradykinesia, hypokinesia, balance and gait disturbances. No overt behavioral signs were reported in the available atrazine studies. This contrasts with rotenone, which causes a Parkinson

- like syndrome, and r esults in reduced mobility, flexed posture, rigidity and even catalepsy in the rat. Rodriguez (2005) provided some evidence for increased locomotor activity following d

- amphetamine sulfate injection of rats treated with atrazine. However, the authors’ ass ertion that the increased locomotor activity is associated with striatal dopamine deficiency from atrazine treatment should be further explored and supported with a study design that includes L

- dopa and dopamine agonist and antagonist administration.

Sev eral in vitro studies addressed the putative mechanism of dopaminergic toxicity of atrazine, primarily in striatal slices and striatal synaptosomes and vesicles (Hossain and Filipov 2008; Filipov et al. 2007). The results revealed that atrazine, in vitro

, alters striatal dopamine levels possibly by interfering with the vesicular storage and/or cellular uptake of dopamine. Tyrosine hydroxylase per se is not a target of atrazine. The relevance of these in vitro data to dopaminergic mechanisms in vivo is not clear given the high concentrations of atrazine examined in vitro and the complex interactions between various neurotransmitter and endocrine systems in the intact brain. Furthermore, as mentioned above, the available data indicate that the neurot oxicity of atrazine is not specific to dopaminergic systems. The effects of atrazine on aromatic amino acid decarboxylase, monoamine oxidase and neuronal mitochondrial function are unknown.

The somatostatinegic system in mouse brain was used as a mode l by Giusi et al. (2006) to study the impact of atrazine exposure on this system. The rationale for this study was that somatostatin plays a role in growth hormone (GH) secretion and the gender

based differences in GH secretion in rodents. The authors th eorized that the differential in GH secretion may be linked to gender

- related differences in the transduction of somatostatinergic mechanisms in brain and/or pituitary targets. As a result, the researchers concluded that the GH

- and SST

- dependent relation ships “might provide further insights into the types of neurotoxicological mechanisms involved in the delay of the estrous cycle or the alteration of the hypothalamic

- hypophyseal

- gonadal axis in animals treated with atrazine (Wetzel et al. 1994).” Though the authors reportedPage 104 of 210 sexually dimorphic changes in the hypothalamus of females and changes in the regulation of somatostatin receptors in the hypothalamus of both males and females, the reliability of this study is uncertain given the numerous deficiencies (including poor description of the methods and poor quality of micrographs used to substantiate conclusions) identified in the study design and described in the summary review in Appendix A.

A neurobehavioral study in mice is also available for considera tion. In this study Belloni and coworkers (2007) report that atrazine exposure from GD14

- PND21 at low doses (1 or 100 μg/kg/day) led to behavioral effects including delays in righting reflex, decreased temperature, changes in pup vocalizations and an acce leration of the ontogeny of the forepaw grasp response. It should be noted that this study also had a number of deficiencies such as inappropriate statistical analyses and unexplained discrepancies in the sampling of pups from litters which are described in the summary review in Appendix A that call the validity of the results into question.

More closely related to the postulated MOA for atrazine is the research conducted by Cooper et al ., 2007 and Foradori et al. (2009) on the pulsatile release of GnRH neuronal activation after atrazine exposure. The research by Foradori and coworkers show that a brief (4 day) exposure to atrazine at doses ≥ 50 mg/kg/day resulted in a decrease in GnRH activation as indicated by a decrease in cFos activity concomitant to a significant reduction in the LH and FSH surges. Thus it appears that atrazine can induce changes in these hypothalamic neurons. These data, therefore, illustrate the close correlation between effects on the central nervous system ( e.g., effects on GnR H activation) and effects on the reproductive system. Whether or not this downregulation of the GnRH neurons are the result of a direct action of atrazine or atrazine metabolites on the GnRH neuron or the result of increased hypothalamic

- pituitary

- adrenal axis are under investigation. Indeed, recent data show that atrazine exposure leads to a rapid increase in corticosterone and progesterone (adrenal steroid hormone) secretions which decrease GnRH secretion (Fraites et al.,2009; Laws et al., 2009).

In s ummary, there are a number of studies indicating that atrazine and or atrazine metabolites may impact different neuronal pathways of the brain. The reliability and validity of findings on the brain somatostatinergic system and on neurobehavior in the

mous e are uncertain due to serious deficiencies of the studies ( i.e. , Guisi et. al. 2006 and Belloni et al. 2007). The recent studies examining changes in the rat brain dopaminergic systems and associated behavioral changes were also found to have significan t deficiencies that also limit their utility for risk assessment purposes. In addition, results of in vivo animal toxicity tests do not support an effect of atrazine exposure on neurological function including Parkinson’s Disease. The observations that th e chlorotriazines may alter GnRH neuronal activity are consistent with the proposed neuroendocrine MOA of atrazine. Previous studies have demonstrated that atrazine can alter dopamine in hypothalamic regions that are important in the regulation of GnRH

se cretion, consistent with the neuroendocrine MOA of atrazine.Page 105 of 210 A.5.1 Belloni V, Alleva E, et al. 2007. Effects of low doses of atrazine on the neurobehavioural development of mice. Ethology Ecology & Evolution 19: 309

– 322.

The authors stated that the aim of their work was to study possible neurodevelopmental effects of perinatal exposure to low, environmentally relevant doses of atrazine in mouse pups. Specifically, the focus was on somatic parameters and a wide range of reflexes to assess sensorimot or maturation.

The authors noted that mice of an outbred Swiss

- derive strain (CD

- 1) were used in the study; no details on age or body weights were provided. Breeding pairs were formed, and on GD 12, males were removed and females housed singly. GD 14 females were assigned to one of three treatment groups: control (received corn oil); low

- dose atrazine (1 μg/kg/day) and high

- dose atrazine (100 μg/kg/day). Atrazine (98.4% purity) was dissolved in corn oil and administered daily to female mice by self administration drinking from a modified syringe. The authors stated that the procedure was not stressful since the animals were previously trained to drink from the apparatus. The treatment period began GD 14 until PND 21. The pups were not treated di rectly and were only exposed by transplacental transfer or via lactation. On PND 2, litters were culled to 8 per litter, 4 males and 4 females.

For behavioral testing, two male and two female pups from 32 different litters were used for postnatal assessm ent of somatic and neurobehavioral development. A total of 128 mice were subjected to behavioral testing between 2 and 15 days of age. Assessment of somatic growth and sensorimotor development included the following parameters: righting reflex, cliff av ersion, forepaw grasp, and auditory startle. Only brief descriptions of these tests were provided. Scoring was as follows: 0, no response; 1, uncertain; 2, incomplete; and 3, full. On PND 2, 4, 9, and 12, pups were weighed.

Evaluation of pup vocalizations (ultrasonic calls) was also performed on PND 4, 9, and 12. The authors provided adequate details on this evaluation. A spectrographic analysis of ultrasonic calls was also performed on PND 9 (which the authors mention is th e peak day in the ontogenetic profile of ultrasonic vocalization). In addition, body temperature was recorded on PND 4, 9, and 12.

The authors reported that there were no differences in dam body weight gain during gestation. A pup body weight decrease of approximately 8% was noted in both atrazine treatment groups compared with the control group on PND 2; no effects were noted at PND 4, 9 and 12. Righting reflex (expressed as “day of full response”) was delayed in the high

- dose atrazine group (mean of 6.60 vs. control mean of 4.60). There was an acceleration of forepaw grasp (“day of full response”) in the low

- dose atrazine group (mean of 5.18) and high

- dose atrazine group (mean of 5.25) in comparison with the control group (mean of 7.50).

On PND 9, mean body weight temperatures were significantly lower in the males and females of the low

- dose atrazine group (5% decrease in males; 4% decrease inPage 106 of 210 females) when compared with controls. There were no significant changes on PND 4 and 12 and no significan t changes in the high

- dose group.

Reviewer’s Comments: The authors indicated that were was a tendency towards a decrease in the mean number of ultrasonic calls in atrazine

- treated pups when compared to controls on PND 9. However, the data (in contrast to controls) show a decrease (not statistically significant) in atrazine

- treated females (both low and high dose groups) and only the low

- dose group for atrazine

- treated males. Moreover, the decreases in mean number of ultrasonic calls in atrazine

- treate d females on PND 9 did not display a clear dose

- response relationship. Based on spectrographic analyses of the ultrasonic calls, the authors claim that there was a tendency towards a longer ultrasonic call mean interpulse (seconds) in atrazine

- treated ani mals compared to control groups. However, the data indicate that this tendency was only apparent in the low

- dose atrazine group of females or when data from males and females were combined. No differences were noted in the high

- dose atrazine groups, whet her data are presented as sex specific or combined. Peak frequency was slightly (not statistically significant) increased in atrazine

- treated males (low and high dose groups) and in atrazine

- treated females (just the high dose group).

The authors concl uded that exposure to low doses of atrazine during the fetal and suckling period interferes with some aspects of somatic growth of pups in a reversible manner, and more clearly with their neurobehavioral development. However, this study has serious defici encies and flaws. The primary problem entails the inappropriate sampling of pups from litters and the statistical evaluation of the data. The authors fail to account for the potential effects of litter, and the key results reported by the authors rely o n only individual pup measurements which inflates sample size. The failure to account for litter effects can result in increased type 1 errors and overestimate any effects, thus reducing the validity of the findings of the study.

In addition to inappr opriate statistical analyses, there were unexplained discrepancies in the sampling of pups from litters. For instance, a lower number of pups were evaluated for somatic growth and sensorimotor development, body temperature, and ultrasonic call emittance c ompared to the actual number of pups available. For the spectrographic analyses of ultrasonic vocalization, not only were significantly less animals evaluated in contrast with the assessment of mean ultrasonic calls, but the reported total numbers of pups evaluated did not match the total reported for males and females separately. The number of animals evaluated for spectrographic analyses of the ultrasonic calls should match the numbers evaluated for the mean number of ultrasonic calls.

Other deficienc ies of the study include a lack of details on the procedures used for assessment of somatic growth and sensorimotor development, a lack of inclusion of positive controls that would support the reliability of the methodology, the lack of objective and valid ated behavioral tests, lack of neuropathology, and the lack of actual spectrograms to corroborate the spectrographic results. The serious deficiencies of these studies raise questions about the validity and reliability of the data and its use for risk ass essment purposes.Page 107 of 210 A.5.2 Coban A, and Filipov, N M. 2007. Dopaminergic toxicity associated with oral exposure to the herbicide atrazine in juvenile male C57BL/6 mice. Journal of Neurochemistry 100: 1177

- 1187.

The objectives of this study were to de termine the following: (1) the effects of atrazine exposure on striatal neurochemistry and striatal expression of tyrosine hydroxylase; (2) whether atrazine effects on striatal neurochemistry are associated with dopaminergic neuron loss; and (3) whether t he neuronal loss is permanent and specific to the substantia nigra pars compacta.

One

- month

- old male C57BL/6 mice were administered atrazine (5, 25, 125, and 250 mg/kg/day) by daily gavage for 14 days. Several indices of neurotoxicity were evaluated on days 15, 22 or 64. Dopamine and its metabolites, 3, 4

- hydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured by high performance liquid chromatography. Striatal tyrosine hydroxylase levels were determined by western

- blot analysis. Fixed half

- brains were used to analyze neurons in the substantia nigra pars compacta and ventral tegmental area by immunohistochemistry/unbiased stereology.

No effects of atrazine treatment on body weights were reported (data not shown). Atrazine at do ses of 125 and 250 mg/kg decreased (dose dependently) striatal dopamine (approximately 80 ng/mg protein at 250 mg/kg vs. 100 ng/mg protein in controls), DOPAC (approximately 6 ng/mg protein at 250 mg/kg vs. 5 ng/mg protein in controls) and HVA (approximate ly 6 ng/mg protein at 250 mg/kg vs. 8 ng/mg protein in controls) levels on day 15. There was no difference between control and atrazine

treated animals in striatal dopamine levels at day 22. Slight decreases in DOPAC and HVA were present at day 22. At d ay 64, there were no statistically significant effects on DOPAC and HVA. There were no effects of atrazine treatment on striatal levels of TH as determined by western blot analysis.

On day 15, there was a decrease in the number of TH

- positive neurons i n both the substantia nigra pars compacta and ventral tegmental area. The loss of neurons was statistically significant at 250 mg/kg/day (the high dose). At day 22, the loss of neurons in the substantia nigra pars compacta was significant at 25 mg/kg/day dose. In regard to the ventral tegmental area, there was a significant decrease in the number of TH

positive neurons at days 14 and 22. The decrease in the ventral tegmental area was reported to be 19% in contrast to a 32% decrease in the substantia nig ra pars compacta. On day 64, atrazine exposure decreased the number of TH

- positive neurons in both the substantia nigra pars compacta and ventral tegmental area, with the overall effect being significant at all dose levels in the ventral tegmental area. The authors reported that at 64 days, the effect on the neurons in the substantia nigra pars compacta at the high dose was only marginally greater than the effect in the ventral tegmental area (33% cell loss in substantia nigra pars compacta vs. 28% cell l oss in the ventral tegmental area).Page 108 of 210 Reviewer’s Comments: There are several limitations with this study:

The reason for a lack of a dose

- response relationship with respect to the number of TH+ neurons is not clear. The study did not include a positive control such as 6

- hydroxydopamine which produces dose dependent alterations in dopamine and dopamine metabolites and loss of tyrosine hydroxylase

- immunoreactive cells in the substantia nigra. The results reported for the atrazine

- induced TH+ neuronal cell numbers (only bar graphs presented) cannot be corroborated since there was no presentation of photomicrographs of brain sections to correlate with the findings presented in the bar graphs. A lso, there was no information on the calibration of the stereological test system. The only statement on the precision of the estimate of neuronal counts was the calculation of a Gundersen coefficient of error. No histological examination of the substant ia nigra and ventral tegmental area was performed; this would provide insight into any morphological alterations to support their cell number data. The dose

- response relationships with regard to dopamine and dopamine metabolite levels were flat. Also, t he decreases were slight even at the two highest doses tested ( i.e. , 125 and 250 mg/kg/day). A positive control, for example, 6

- hyroxydopamine (discussed above) was not included in the study. There were no functional assessments of the dopaminergic altera tions, for example, assessment of behavior and motor activity to correlate with the neurochemical findings. Neurochemical analyses were not performed for the ventral tegmental area which would have provided some support for the observed findings of the los s of dopaminergic neurons in this region.

Coban and Filipov also presented data for days 22 and 64 of the experiment revealing only slight changes in the dopamine metabolites DOPAC and HVA at day 22 (flat dose response) and no alterations in dopamine and its metabolites at day 64. However, these data ( i.e. , Figure 3 on page 1182 of the study) are contradicted by the description of the data in which the authors state that the “some of the animals received MPTP on day 15” (no further details provided). MP TP has been associated with a Parkinsonian

like syndrome, and it is not certain if the word MPTP was a typographical error, if MPTP was meant as a possible positive control (not discussed in methods or any other section) or if the wrong results were presen ted. If MPTP was included as a positive control it is not clear why it was not administered to mice at the same treatment start time as atrazine.

A.5.3 Filipov N M, Stewart M A et al. 2007. Dopaminergic toxicity of the herbicide atrazine in rat striat al slices. Toxicology 232: 68

– 78.

This in vitro study examined the effects of atrazine treatment of various aspects of dopamine metabolism in rat striatal slices. Striatal tissues from adult male Sprague

Dawley rats were incubated with up to 500 μM atrazine for 4 hours. The following were evaluated: tyrosine hydroxylase activity using L

- DOPA accumulation as an indicator, high potassium

- stimulated dopamine release, media dopamine levels in the presence ofPage 109 of 210 the dopamine uptake blocker nomifensine, and striatal dopamine metabolism. In addition, the effects of in vitro exposure to atrazine on LDH levels was performed to determine if any potential dopaminergic effects are caused by non

- specific cytotoxicity. Dopamine and metabolites, 3,4

- hydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) were measured.

Atrazine treatment at concentrations of 100 μM or greater resulted in a dose

- dependent decrease in tissue levels of dopamine. At 50 μM or greater concentration of atrazine, DOPAC + HVA/DA rat io was dose

- dependently increased. Tyrosine hydroxylase protein levels and activity were not affected by atrazine treatment. However, atrazine decreased high potassium

- induced dopamine release; the increase in media dopamine in the presence of nomifensi ne was increased even further by atrazine in a dose

dependent manner. These effects were observed at levels that were not toxic to the tissue. The authors concluded that their results suggest that atrazine decreases tissue dopamine levels not by affectin g tyrosine hydroxylase activity, but possibly by interfering with the vesicular storage and/or cellular uptake of dopamine.

Reviewer’s Comments: The results revealed that atrazine, in vitro , alters striatal dopamine levels possibly by interfering with the vesicular storage and/or cellular uptake of dopamine. Tyrosine hydroxylase per se is not a target of atrazine. The relevance of these in vitro data to dopaminergic mechanisms in vivo is not clear given the high concentrations of atrazine examined in vit ro and the complex interactions between various neurotransmitter and endocrine systems in the intact brain. The effects of atrazine on aromatic amino acid decarboxylase, monoamine oxidase and neuronal mitochondrial function are unknown.

A.5.4 Foradori, C D, Hinds, L R, et al. 2009. Atrazine inhibits pulsatile luteinizing hormone release without altering pituitary sensitivity to a gonadotropin

- releasing hormone receptor agonist in female wistar rats. Biology of Reproduction. 81: 40

– 45.

The objecti ves of this study were to examine the effects of atrazine on normal tonic release of luteinizing hormone (LH) and to elucidate the site of action of atrazine in the hypothalamic

- pituitary

- gonadal axis. To assess potential effects of atrazine on pulsatile LH release, ovariectomized adult female Wistar rats were administered atrazine (50, 100, or 200 mg/kg/day by gavage) or vehicle (1% carboxymethylcellulose sodium salt) control for 4 days. At termination, blood samples were obtained using an indwelling rig ht atrial cannula (implanted on second day of treatment). Samples were examined for LH levels. There was a significant reduction in LH pulse frequency and a concomitant increase in pulse amplitude in the 200 mg/kg/day group.

The second experiment invo lved validation of a protocol for GnRH immunoneutralization and pituitary response to GnRH agonist. Utilizing the same experimental procedure above, animals were administered GnRH antiserum via cannula to block the actions of endogenous GnRH. The anti

- Gn RH serum was prepared against keyhole limpet hemocyanin

- conjugated GnRH. Following blood sampling, animals were treated withPage 110 of 210 one of four doses of GnRH receptor agonist, D

- Ala 6

- des

- Gly

- NH 2 10

- GnRH

- ethylamide (0.01, 0.1, 1.0, or 10 ng; 4

- 6 animals per treatm ent group). LH levels were measured. The results showed that LH levels were at or below the detection level of the assay in animals administered the GnRH antiserum, indicating that the effect of endogenous GnRH on the pituitary release of LH was suppress ed. Administration of GnRH agonist resulted in dose

- dependent increases in mean plasma LH levels.

The third experiment entailed an examination of the effects of atrazine on in vivo pituitary response to GnRH agonist. The objective was to determine wh ether atrazine reduces pituitary sensitivity to a GnRH agonist. Ovariectomized rats were administered atrazine (0, 50, 100, or 200 mg/kg/day by gavage) for 4 days. On the second day of treatment, intra

- atrial cannulae were implanted, and rats were treate d with anti

- GnRH serum via cannula. On the last day of treatment, sampling of blood was performed and D

- Ala 6

- des

- Gly

- NH 2 10

- GnRH

- ethylamide (0.1 or 1.0 ng) was administered via catheter. Sampling was continued for an additional 90 minutes. Atrazine

- treat ed animals that had been immunoneutralized with the anti

- GnRH serum showed an LH response comparable to that observed in the control group. Both doses of D

- Ala 6

- des

- Gly

- NH 2 10

- GnRH

- ethylamide caused an increase in the circulating concentration of LH. Howe ver, there was no significant change in the levels of LH related to atrazine treatment.

The authors concluded that although LH pulse period and amplitude were increased in atrazine

- treated animals, the anterior pituitary function does not seem to be alter ed by atrazine, as LH levels were not reduced in atrazine

- treated animals in response to a GnRH agonist after GnRH immunoneutralization. Additionally, the inhibition of LH pulses and the disruption of cyclicity in female rats treated with atrazine are lik ely to be mediated by interference with central mechanisms controlling GnRH release from the hypothalamus. The brain appears to be the primary target mediating the effect of atrazine on LH release in the rat.

Reviewer’s Comments: The Foradori study pr ovides added support for the hypothesis that atrazine targets the brain to modify the neuronal control of pituitary function. Evaluating the secretion of gonadotropin releasing hormone (GnRH) in rats revealed that atrazine alters the release of this neurop eptide from the brain. GnRH is essential for LH release from the pituitary.

A.5.5 Foradori, C D, Hinds, L R, et al. 2009. Effects of atrazine on GnRH neuroendocrine function after its withdrawal in the adult female wistar rat. Biology of Reproduction 81(6): 1099

– 1105.

The authors previously reported that atrazine treatment decreased LH pulse frequency and increased pulse amplitude in ovariectomized rats without changing pituitary sensitivity to a GnRH receptor agonist. The authors interpreted the se results to suggest a neural site of atrazine’s action. In order to explore this possibility, the authors examined the effect of atrazine on the estrogen and progesterone

- induced LH surge in freely moving, ovariectomized rats to determine if atrazine’s inhibition of LH secretion coincides with a reduction in GnRH neuronal activation. The immediate early genePage 111 of 210 product FOS (cFos) was colocalized in GnRH neurons as a measure of cellular activity. In addition, withdrawal studies were performed to determine whether LH levels and measures of GnRH neuronal activation would return to control levels following the termination of atrazine treatment.

To examine the potential effects of atrazine on GnRH neuronal activation, ovariectomized young adult female Wistar rats were treated (by gavage in 1% carboxymethylcellulose sodium salt) once daily with atrazine (0, 50, 100, or 200 mg/kg) for 4 consecutive days, and were primed with 17beta

- estradiol

- 3

- benzoate and progesterone (EB + P) to induce LH surge. Near the pe ak of the predicted LH surge, animals (n=4

- 6) were anesthetized and perfused, and brains were removed. Brains were processed for GnRH and cFOS using immunohistochemistry. To determine if atrazine alone would have a stimulatory effect on GnRH neuronal act ivation, a second cohort of animals was treated similarly except that no hormones were given to induce GnRH and LH surges. For this part of the experiment, rats were treated with 50 mg/kg atrazine or vehicle for 4 days. To examine the effects of atrazine withdrawal on hormone

- induced LH surge and GnRH activation, ovariectomized animals were given a 4

- day regimen of atrazine (200 mg/kg) by gavage. Control animals received vehicle only. An LH surge was induced in these animals either during the atrazine t reatment period or 2 days or 4 days after the cessation of atrazine treatment.

Atrazine treatment inhibited the EB+P LH surge at all doses. Atrazine also reduced the area under the curve (AUC) for the LH surge at all doses examined. Atrazine treatmen t did not affect peak FSH levels; however, atrazine treatment at 200 mg/kg reduced the AUC for FSH levels in this group. Treatment with 100 mg/kg and 200 mg/kg atrazine reduced the percentage of GnRH neurons immunoreactive for cFOS compared with the vehic le

- treated and 50 mg/kg atrazine groups. There was no difference between the 100 and 200 mg/kg atrazine groups. Treatment with 100 mg/kg and 200 mg/kg atrazine also reduced the mean number of GnRH neurons immunoreactive for FOS; there was no difference between these groups. The results also indicated that atrazine alone does not induce FOS immunoreactivity in GnRH neurons. Within 4 days (but not 2 days) after cessation of atrazine treatment, the reductions in GnRH activation and hormone

- induced LH surg e returned to control levels.

The authors concluded that their data indicate that atrazine affects neuroendocrine function in the female rat by actions at the level of the GnRH neuron and that the acute effects of high doses of atrazine can be reversed within 4 days after withdrawal of treatment. The authors acknowledge that “it remains unclear whether atrazine acts directly on GnRH neurons or on one of many neurotransmitter containing neurons in the brain that are known to have a role in controlling t he preovulatory surge of GnRH.”

Reviewer’s Comments: This study provides further support to their previous findings (Foradori et al. 2009).Page 112 of 210 A.5.6 Giusi G, Facciolo RM, et al. 2006. The endocrine disruptor atrazine accounts for a dimorphic somatostati nergic neuronal expression pattern in mice. Toxicological Sciences 89(1): 257

– 264.

The intent of this study was to examine the potential effects of atrazine on various somatostatin receptor subtypes (in particular, SST 2, 3, 5 ) including possible regio nal specificity in some hypothalamic and extra

- hypothalamic estrogen

- enriched brain regions. The authors provided a rationale for the selection of the somatostatin receptor subtypes. The authors noted that the widely distributed SST 2, 3, 5 appear relati vely early during development but also remain stable during adult life and exert a key role in the organization of the central nervous system. The authors also noted the role of somatostatin in the control of the secretion of growth hormone (GH) and the s triking differences in GH release between male and female rodents. The authors stated that differentiated GH secretion may be due to gender

- related differences in the transduction of somatostatinergic mechanisms in brain and/or pituitary targets. Finally , the authors concluded that the GH

- and SST

- dependent relationships “might provide further insights into the types of neurotoxicological mechanisms involved in the delay of the estrous cycle or the alteration of the hypothalamic

- hypophyseal

- gonadal axis i n animals treated with atrazine (Wetzel et al. 1994).” The interest in regional specificity was based on the observations that altered SST levels have been reported for hypothalamic nuclei.

Outbred Swiss

- derived strain (CD

- 1) mice were used in the study . Gestation day (GD) 14 females were divided into 2 groups: one group (low

- dose) received 1ug/kg day atrazine via drinking water from a syringe, while the second group (high

- dose) received 100 ug/kg day atrazine. The authors noted that the procedure was monitored to make sure that pups did not attempt to drink the solution from the syringe. Treatment period was from GD14 to postnatal day (PND) 21. On PND 21, mothers were removed and F1 mice of both sexes (n = 6 for both sexes) continued with the treatm ent protocol (control group was included). At PND 60

– 65, the animals were sacrificed and brains removed. Brain sections were prepared. Amino cupric silver staining was used for examining potential degenerative changes. Reverse transcription polymerase chain reaction was applied for the detection of SST 2,3,5 . In situ hybridization was utilized for mRNA.

The authors reported a high

- dose atrazine

- dependent dimorphic neurodegeneration pattern based on the amino cupric silver staining results. The aut hors described the neurodegeneration as “a consistently evident argyrophilic reaction of axonal processes and perikarya in some extra

- hypothalamic areas, specifically the external pyramidal neuron layer of the female hippocampal area, but not those of the male.” The authors also indicated that examination of another hippocampal field ( i.e. , the dentate gyrus), the neurons showed a rather significant overall reduction in the total number of neuronal cells accompanied by significant perikaryal swelling plus well

- developed astrocytic formations in the female. The dimorphic pattern was also evident in the hypothalamus, as demonstrated by significantly darker axonal processes in the female arcuate hypothalamic nucleus. The dimorphic pattern was not observed i n extra

- hypothalamic areas such as the cortex or striatum.Page 113 of 210 In the high

- dose female group, the authors reported a very strong (p < 0.001) upregulation of sst2 expressing neurons in the female hypothalamic areas, specifically the suprachiasmatic nucleus (+110%) and ventromedial hypothalamic nucleus (+65%), whereas a similar downregulatory trend was noted in the some extrahypothalamic areas such as the striatum (

- 96%) and central amygdaloid nucleus (

- 45%). In the high

- dose male group, the authors report ed very strong upregulatory and downregulatory actions for sst3 expressing neurons, with upregulation in the arcuate hypothalamic nucleus (+120%) and basolateral amygdaloid nucleus (+78%), and downregulation in the cortical areas (

- 88%) and dendate gyrus (

- 105%).

The authors concluded that the observed neurotoxicological effects of atrazine are “very likely linked to dimorphic expression patterns of specific somatostain subtypes in discrete but key hypothalamic and extrahypothalamic areas” of the mous e.

Reviewer’s Comments:

**•** There was a lack of details on the age at which mice were bred, the source and purity of atrazine, and the body weights of the animals, and systemic toxicity measurements were not performed. It is not clear if atrazine was dis solved in corn oil which was administered to the controls. It is also not clear if the treated animals received the same amount of solution ( i.e. , 0.1 ml) as was administered during a pretreatment period to train the mice to drink from a syringe.

**•** Dams were treated with atrazine during gestational and lactational phases, however, there was no mention of standardizing litter sizes. Standardization of litter size would reduce between

- litter variability.

**•** Also, several of the reference citations did n ot match or support statements presented elsewhere in the study. For example, the reference to Surrales (1995) pertains to alachlor, mitocyn, and vinblastine. Gould et al. (1997) refers to research on polychlorinated biphenyl mixtures, not atrazine.

**•** The f ew photomicrographs of the amino cupric silver staining results utilized to demonstrate critical neuronal degeneration were of very poor quality and not presented in color to portray the reactive staining, and were based on a small sample size, and only re presentative sections were provided. A “blind” evaluation was not performed. There was no presentation of count data (all stained reactive cells within an area) with statistical analyses to quantify the results of the reactive staining. Furthermore, no cell number data (with appropriate statistical analyses) were provided to support the claim that there was “a rather significant overall reduction in the total number of neuronal cells...”. Their claim cannot be confirmed without these data.

**•** Only one re presentative photo on reverse

- transcriptase polymerase chain amplification results was provided, and there was no mention if there was a control for possible genomic contamination in RNA preparations.

**•** No rational basis to explain the purported differentia l neuronal expression and dimorphic sex effects was offered. For example, striatal neuronal degeneration was reported in both males and females (although the photomicrographs were too poor in quality to verify these findings), yet downregulation ofPage 114 of 210 somato statinergic receptor subtype 2 (sst2) was noted only in the females. In other brain areas of the female there was upregulation of sst2. There were similar difficulties in correlating neuronal degeneration with differential expression of sst subtypes and dimorphic sex distribution in other brain regions, and no plausible biological mechanism to explain the pattern of effects was discussed. Moreover, the relevancy of these findings is not clear.

**•** The study did not address the potential confounding by cortic osterone

. Based on the number of serious limitations and no other corroborative support in the literature, the preliminary results of this study are inconclusive.

A.5.7 Hossain M M and Filipov N M. 2008. Alteration of dopamine uptake into rat striat al vesicles and synaptosomes caused by an in vitro exposure to atrazine and some of its metabolites. Toxicology 248: 52

– 58.

To better clarify the potential mechanisms of atrazine

- mediated effects on striatal dopamine, this study examined the effects o f atrazine on the vesicular and synaptosomal uptake of striatal dopamine, which may result in disrupted vesicular storage and/or cellular uptake of dopamine. The study design involved in vitro atrazine exposure using isolated rat striatal synaptosomes an d synaptic vesicles. Besides atrazine, effects of atrazine metabolites, i.e. , didealkly atrazine (DACT), desethyl atrazine (DE) and desiopropyl atrazine (DIP) were also investigated. Limited details were provided on the various methods such as preparatio n of synaptic vesicles and striatal synaptosomes, vesicular uptake of dopamine, and synaptosomal uptake of dopamine.

Atrazine (1

– 250 μM) inhibited dopamine uptake into synaptic vesicles, and the inhibition was dose dependent. DACT did not affect vesicu lar dopamine uptake, while DE and DIP significantly decreased vesicular dopamine uptake. Kinetic analysis (nonlinear isotherm regression) of the vesicular dopamine uptake showed that atrazine decreased the V max parameter but not K m .

Atrazine and DE inc reased (6

– 13%) synaptosomal dopamine uptake; DACT and DIP were without effect. Atrazine, DE and DIP increased the ratio of synaptosomal/vesicular uptake of dopamine. The authors concluded that atrazine and two of its metabolites (DE and DIP) decrease s triatal dopamine levels, at least in part, by increasing cytosolic dopamine, which is prone to oxidative breakdown. The authors note that the effects of atrazine on aromatic amino acid decarboxylase, monoamine oxidase and neuronal mitochondrial function r emain unknown. Previously, the authors (Filipov et al. 2007) showed that atrazine does not alter the function of tyrosine hydroxylase substantially.

Reviewer’s Comments: The results revealed that atrazine, in vitro , alters striatal dopamine levels possi bly by interfering with the vesicular storage and/or cellular uptake of dopamine. Tyrosine hydroxylase per se is not a target of atrazine. The relevance of these in vitro data to dopaminergic mechanisms in vivo is not clear given the highPage 115 of 210 concentrations of atrazine examined in vitro and the complex interactions between various neurotransmitter and endocrine systems in the intact brain. The effects of atrazine on aromatic amino acid decarboxylase, monoamine oxidase and neuronal mitochondrial function are unknown.

A.5.8 Rodriguez V M, Thiruchelvam M, Cory

- Slechta D A. 2005. Sustained Exposure to the widely used herbicide atrazine: altered function and loss of neurons in brain monoamine systems. Environmental Health Perspectives. 113(6): 708

- 715.

Th e purpose of this study was to examine the hypothesis that atrazine adversely affects brain dopamine systems, in particular, the nigrostriatal and mesocorticolimbic pathways. In addition, the underlying mechanisms of any potential changes in dopamine func tion in these pathways were explored by examining the acute effects of atrazine on striatal dopamine release.

In the chronic phase of the study, 30 male Long

- Evans rats (9 months of age) were exposed via the diet to 0, 5 or 10 mg/kg/day atrazine for 6 m onths. Body weights and food consumption were monitored throughout the study. Locomotor activity was recorded at 2, 3, and 6 months of atrazine treatment and 2 weeks after cessation of treatment. At the 2

- month time point, locomotor activity was measure d on 3 consecutive days, in which the rats were injected saline intraperitoneally 5 minutes before the session during the first 2 days, and an injection of 1 mg/kg d

- amphetamine sulfate on day 3. Only a single locomotor activity session was performed at t he 3

- and 6

- month time points and at 2 weeks after termination of treatment. Locomotor activity was recorded during the light phase for 7 hours. The hypothalamus, prefrontal cortex, nucleus accumbens, and the striatum were removed from sacrificed animals 2 weeks after termination of the treatment. Monoamine and monoamine metabolites were measured by HPLC with electrochemical detection. Immunohistochemical and unbiased stereologic analyses of tyrosine hydroxylase from the substantia nigra pars compacta a nd ventral tegmental area were performed. Microdialysis was used to evaluate changes in striatal dopamine release after acute i.p. exposures to 100 or 200 mg/kg atrazine in sessions lasting 7 hours. Animals were subjected to stereotaxic surgery and cannu lation. Brain histology was performed on the rats at the completion of micordialysis sampling.

No treatment

- related changes in body weights or food consumption were reported (data were not shown). Rats treated for 2 months with 10 mg/kg atrazine exhibit ed an enhanced horizontal locomotor activity response (70% relative to the 0 and 5 mg/kg groups) to d

- amphetamine. The increased locomotor activity persisted at the 3

- and 6

- month time periods, as well as 2 weeks after treatment, time periods carried out in the absence of d

- amphetamine administration. At 2 weeks after treatment with atrazine, locomotor activity was 40% above control values. A statistically significant decrease (approximately 20%) in striatal dopamine levels at 10 mg/kg/day was noted. D opamine levels were increased in the prefrontal cortex in an inverse U

- shaped fashion; the increase at 5 mg/kg/day was 30

– 40%, while levels declined towards control levels atPage 116 of 210 10 mg/kg/day dose. Significant decreases (10

– 15%) in hypothalamic serotonin level were observed at the 5

- and 10

- mg/kg/day doses. The 10 mg/kg/day dose was associated with a significant decrease (15

– 20%) in norephineprine levels in the prefrontal cortex. The authors reported no changes in the metabolites of serotonin or dopami ne or dopamine turnover in any region.

At 2 weeks after the 6

- month treatment period, the number of tyrosine hydroxylase positive (TH+) and tyrosine hydroxylase negative (TH

- ) cells were reduced in the substantia nigra pars compacta and ventral tegmental areas. This effect was primarily due to the 10 mg/kg/day dose level. Mean reductions in cell numbers ranged from 9 to 13% in the 10 mg/kg/day group, whereas those in the 5 mg/kg/day group ranged from 0 to 3%.

Following an acute i.p. administra tion of 100 or 200 mg/kg atrazine, a significant decrease in basal dopamine release was noted as measured at 90, 120, 150, and 180 minutes postadminstration. By 150 minutes, the decreases averaged approximately 40% and were seen in both dose groups. The authors also noted a dose

- dependent decrease in potassium

- stimulated dopamine release. No changes in the dopamine metabolites (DOPAC or HVA) were observed in the microdialysis component of the study.

The authors concluded that there findings “demonstr ate that atrazine may have broad effects on brain monoamine systems and thereby influence a wide range of behavioral functions.” The authors acknowledge that “additional studies are needed to unravel the targets of atrazine and the mechanisms of its effec ts, as well as the ultimate human health consequences of such exposures for behavioral and/or neurological dysfunctions.”

Reviewer’s Comments: This study has a number of limitations, including:

**•** No photomicrographs were provided to support and corrobora te the findings of stereological examination of TH+ and TH

- cell loss (only cell number data were provided), and no histological examination to describe actual morphological changes was discussed. There was no information on the calibration of the stereol ogical test system and no information on control for potential sources of variability in the stereological estimations of cell numbers. There was no assessment of the precision of the estimations of neuronal counts.

**•** Behavioral assessment was limited to d

- amphetamine

- induced locomotor activity; inclusion of dopamine agonists and antagonists and L

- Dopa in the experimental design would help support the findings.

**•** The study did not include a positive control such as 6

- hydroxydopamine which produces dose depe ndent alterations in dopamine and dopamine metabolites and loss of tyrosine hydroxylase

- immunoreactive cells in the substantia nigra.Page 117 of 210 A 5.9 Solari, M, Paquin, J, Ducharme, P, Boily, M (2010). P19 neuronal differentiation and retinoic acid metabolism as c riteria to investigate atrazine, nitrite, and nitrate developmental toxicity. Toxicol Sci 113 (1):116

- 126.

In an in vitro study using neuronal differentiation as an indicator of developmental toxicity, Solari et al. (2010) exposed cultured mouse P16 emb ryonic carcinoma cells to atrazine along with NaNO 3 and NaNO 2. The basis of the assay is that P16 cells, which can differentiate into neurons in response to retinoic acid (RA), are sensitive to agents that can influence all trans RA (atRA, the active meta bolite of vitamin A) metabolism and cause developmental toxicity since RA is an essential micronutrient for growth and embryonic development. Consequently, undifferentiated P16 cells, grown in differentiation culture medium, were treated with 0, 500, 1000 or 2000 μg/L of atrazine, prepared in dimethyl sulfoxide in the presence and absence of 0.05 μM all

- trans RA (at

RA). Cells were allowed to aggregate for 4

- 5 days and differential medium, containing atrazine, was replenished at days 2.and 4. After treatm ent, cells were dissociated with trypsin, processed and analyzed for the expression of stage

- specific embryonic antigen

1 (SSEA1, a cell

- surface glycoconjugate marker of the undifferentiated state) and βIII

tubin (a neuron

- specific cytoskeletal protein) us ing immunostaining and flow cytometry. Extracts, prepared .from lysed cells, were subjected to gel electrophoresis for confirmation of gene expression using Western blot analysis. Cell viability and proliferation were also determined as well as retinoid a nd protein content. The limited data that were available for atrazine suggest that no adverse effects on cell viability, cell proliferation or RA metabolism resulted from exposure to atrazine concentrations observed in rivers in Quebec.

Reviewer Comm ents: In general, the study represents the development of a new in vitro assay to detect potential developmental toxicants; thus the major emphasis in the article is on techniques that validate the test system. Since the extent of data on atrazine are lim ited (generally presented as bar graphs) and the highest concentration tested (2 μg/mL) was not cytotoxic, the relevance of these findings to the atrazine risk assessment is questionable.

A.5.10 Belloni V, dessi

- Fulgheri, F, et al. 2010. Early exposure to low doses of atrazine affects behavior in juvenile and adult CD1 mice. Toxicology (2010), doi:10.1016/j.tox.2010.07.002

The authors stated that the aim of the study “was to evaluate effects of perinatal exposure to environmentally relevant subtoxic do ses of atrazine (ATZ), on neurobehavioral development in mice and investigate possible alterations in steroid hormone metabolism.”
Female and male mice delivered from CD1 dams were exposed daily from Gestational Day 14 (GD14) until Postnatal Day 21 (PND2 1) to 1 or 100 μg/kg bw ATZ. Locomotor and exploratory activity, social interactions and cognitive performance were evaluated at PND 16, 31 and 60, respectively. On PND 60

- 65, 10 F1 males (1/litter in each treatment and control group), were randomly sel ected and sacrificed by cervicalPage 118 of 210 dislocation. Liver and testes from control and treated groups were removed and weighed. Testicular parameters (sperm head number and concentration on right testes, histological evaluation of left testes), rate of testos terone metabolism (hepatic microsomal testosterone hydroxylase) and aromatase activity in F1 male liver were analyzed.
The authors reported that there were no clinical signs of toxicity in dams and pups. No significant changes in absolute and relative testicular weights were noted in F1 male mice at PND 60

- 65. A reduction in testicular sperm count and concentration was observed in treated animals. The authors indicated that there were no significant morphological changes of the germinal epithelium in treated animals, although in 1/8 in both ATZ

- treated groups the epithelium organization was altered in some tubules.

I n regard to the open

- field test, the authors concluded that duration of exploratory behavior was increased when compared to controls. For the novel object test, the authors indicated an increase in duration of sniffing and a tendency to perform higher

num ber of contacts with the object in both ATZ

- treated groups compared to control (no differences between sexes).
With respect to social interactions, the authors reported a “significant main effect of treatment” on investigative

- affilative and explorative behaviors in males, in particular, for the low

- dose ATR group there was a higher frequency of elements grouped in “factor 1” (squire, genital sniff, following, and allogrooming) compared to controls. A less pronounced and nonsignificant similar trend w as noted for the high

- dose ATR group. The authors also stated that non

- social exploratory component of “factor 2” (explore, wall rearing, sniff) was affected by the high

- dose ATR treatment, in which exposed animals exhibited a clear cut reduction in the habituation profile when compared to controls.
The results of the passive avoidance test revealed improved performance in the learning session for the high

- dose ATR group compared with the low

- dose ATR and control groups. The authors mentioned that “femal es showed higher latency than males to enter the escape compartment.” Also, no effect of treatment was observed in the retest session, whereas, sex affected the passive avoidance response (females taking longer in stepping

- through than males).

The a uthors reported effects of atrazine on hepatic testosterone hydroxylase and aromatase activities in the liver. They stated that treatment with both ATZ doses produced slight changes in the rate of selected testosterone hydroxylases. The affected isoforms were CYP 2C and 2B, as reflected by the differences in the formation of 16α

and 16β

- hydroxyl

- T in the livers from treated and control animals. The authors also reported a slight decrease in hepatic aromatase activity (statistically not significant) in the treated animals compared with controls. The authors concluded that “developmental exposure to non

- toxic, environmentally relevant doses of ATZ can produce subtle functional alterations” in juvenile and adult mice.

Reviewer’s Comments: This publishe d study was not designed as a GLP compliant study to meet Agency toxicology test guidelines for data requirement, but rather toPage 119 of 210 explore potential effects of atrazine on behavior in juvenile and adult mice. A strong point of the study is that the “litter ” was considered in the design and analyses of the results. The behavioral methods are typical of the methods utilized with measuring social and non

- social behaviors in mice. However, there are several notable limitations of the study:
Dosing

- Dosing of the dams was done via licking the dosing solution from a syringe which is not a common dosing procedure. This procedure assumes that the total dose obtained from the syringe was swallowed. There was also no analytical confirmation of atrazine concent rations of the dosing solution. Only 2 dose levels were examined. The authors acknowledge the “controversy” of nonmonotonic dose

- response relationships associated with “endocrine disruptors.” Examination of more than 2 dose levels would assist in inte rpretation of the findings.

In

- house breeding

- Mice were bred in

- house which means that births were likely spread across multiple days. If this is the case, the authors should have presented a table indicating the number of animals from each group bo rn on each day. The implication is that for testing at very early ages one may not have representative pups from all dose groups.

Blind observations

– There was no mention if the observer was “blind” to treatment which would ensure objectivity. This i s critical for behavioral studies of this nature.

Positive Controls

- The study did not include positive controls which would be helpful in interpretation of behavioral findings, and ensure that the investigators can actually detect chemicals that interfe re with behavior. As an example, the biological significance of exploratory behavior (figure 1), in which changes were of small magnitude and non

dose

- response related is dubious. The same problem is also noted for the reported effects of perinatal atraz ine administration on frequency and duration of sniff object behavior in novelty test on PND16 (figure 2). There was large variability in the data and the findings are of doubtful toxicological significance. This is also apparent with the results of the passive avoidance test in which the reported effect is small and not dose related. In addition, the high

- dose ATZ treated animals exhibited improved performance compared with the low

- dose ATZ and control groups, implying that high dose ATZ animals had bet ter memory and learning performance than the control group. The figures did not distinguish between male and female results although the authors noted significant differences between the sexes in regard to passive avoidance. There were also no statistica l analyses to compare the retest (retention) results from the pre

- multitrial (learning) session. In regard to their findings on passive avoidance, the authors claim that their results are consistent with the findings of rats perinatally treated with bisph enol A (Negishi et al. 2004). However, Negishi et al. (2004) expressed reservation of the effects of bisphenol A on passive avoidance and stated “...such changes were statistically ambiguous because of the large individual differences in the experimental conditions used in this study.” Furthermore, Belloni et al. acknowledge several potential confounders which further complicate the interpretation of the passive avoidance results. Belloni et al. state that “possible effects of development ATZ exposure on shock sensitivity thresholds, that could maskPage 120 of 210 associative learning alterations in treated mice, cannot be dismissed on the basis of our results, since estrogens and estrogenic substances can influence nociceptive sensory processing in rodents (Aloisi et a l., 2002).” Also, they mentioned that “we cannot exclude that ATZ may induce alterations in dams, resulting in changes in maternal cares, and, consequently, in offspring’s altered developmental behavioral trajectories.”
Problems with figure labeling

– The labels associated with a number of endpoints are incorrect. For example, the authors reported an effect of atrazine treatment on “duration” of exploring behavior in the open

- field test, however, the relevant figure (figure 1) indicates “frequency.” Fig ure 1 also depicts “duration” to describe “crossing”, and “rearing”; however, “frequency” is the correct descriptor.

Additionally, the figures did not distinguish between males and females, although the authors often made conclusions based on individual sex.

Testosterone metabolism and aromatase activity

– The reported effects of atrazine on testosterone metabolism and aromatase activity are of doubtful biological significance given the slight and non

- dose

- related changes. In addition, one of the refe rences (i.e., Traina et al. 2003) relating to methodology for examining testicular parameters was not presented in the reference section.

Principal Component Analysis

- The application of principal component analysis (PCA) as an exploratory data analysis of social interaction data is an acceptable technique since behavioral variables may be correlated. However, there are limitations with the use of PCA. Most notably, the authors applied a linear PCA method. Since neurobehavioral data may exhibit non linearity, a nonlinear PCA approach should have been explored. Other significant problems with PCA are that it can fail if the data are

non

- Gaussian or time

- dependent. The authors did not address these potential and likely issues. The results of the li near PCA should be interpreted with caution and may be misleading.

The authors provided little description of how the PCA was performed, e.g., the software used, how transformation was performed (orthogonal or other approach), eiganvalue decomposition a pproach, and the matrix (correlation or covariance) examined. The authors also reported a total variance of 75% following varimax rotation. However, the table of individual factors adds up to 66% and it is not clear if the 66% is a typo, the correct valu e following rotation, or if it represents an unrotated value. Reporting of both unrotated and rotated values would be helpful. It is also unclear if the loading factors presented in Table 3 represent values before varimax rotation or after varimax rotat ion. Once again, it would be helpful to see the results of both which can aid in interpretation of the results. It is interesting to note that even with varimax rotation, the PCA analyses cannot explain all of the information, which raises questions abou t the applicability of linear PCA methodology. Finally, it would have been useful if the authors had applied a statistical analyses of the data separate from the PCA analyses for comparative purposes given that the assumptions of the PCA may be flawed.Page 121 of 210 In summary, this study has a number of significant limitations that limit its use for quantitative risk assessment purposes. There are no other studies that have validated the findings presented by Belloni et al.

A.6 IMMUNOTOXICITY

Summary: Immunotoxicity

In the Agency’s 2003 evaluation of atrazine, the immune system was identified as one of the target systems for atrazine toxicity. Since then, numerous studies both in vivo and in vitro have been conducted to further investigate atrazine’s potential to cause immunotoxicity. The in vivo studies reported various potential immunological effects of atrazine on cellular and humoral immune systems. These included natural killer (NK) cell function, delayed

- type hypersensitivity responses to bovi ne serum albumin (BSA), macrophage phagocytic activity, antibody responses to sheep erythrocytes or keyhole limpet hemocyanin, T cell proliferation and cytolytic activity, spleen and thymic cell phenotypes, humoral response after immunization with heat

- kil led Streptococcus pneumoniae , lymphocyte populations in peripheral blood mononuclear cells, and expression of MHC Class II proteins on splenocytes. One study design included a host resistance test, which assesses the function of the immune system in toto (Karrow et al. 2005).

All of the adult in vivo immunotoxicity studies focused primarily on cellular and humoral systems. Several studies evaluated limited systemic effects such as spleen and thymic weights and body weights. None of the studies provided detailed and thorough histological examinations of lymphoid organs(e.g., spleen, thymus, bone marrow, lymph nodes) that would be useful in understanding the potential mechanisms of immunotoxicity. Consistent effects (e.g., decreased splenic and thymic we ights and alterations in splenic and thymic cellularity and phenotypes) were noted across the adult immune studies that examined these endpoints. In many instances, the changes were slight (albeit statistically significant), and given the functional reser ve capacity of the immune system, it is difficult to extrapolate the rodent findings to human health risk assessment. However, it is interesting to note that the results of the host resistance test to validate the biological significance of changes in cell ular and humoral immune function revealed significant decreases in resistance to the B16F10 tumor challenge at doses of 250 and 500 mg/kg/day atrazine, but not at 25 mg/kg/day. Host resistance assays are often considered to be the most relevant test for validating other methods and for extrapolating the potential of environmental agents to alter host susceptibility in the human population.

It is well known that alterations in corticosterone levels may result in impaired immune function through the acti vation of the HPA axis. Pruett et al. (2003; 2009) and Schwab et al. (2005) exposed rats intraperitoneally to atrazine at high doses ranging from 100

- 300 mg/kg in an attempt to determine if corticosterone area under the curve could be used to predict im munotoxicity. The effect of HPA axis perturbations on immunological parameters can be quantified by relating immunosuppression to change inPage 122 of 210 corticosterone levels represented by the area under the corticosterone vs. time curve (AUC) (Schwab et al. 2005). P ruett et al. (2003, 2009) and Schwab et al. (2005) evaluated atrazine in the corticosterone response model including acute and 28

- day exposures to atrazine. In this model, atrazine treatment resulted in a number of immune system effects (e.g., suppressed white blood cell counts, increased neutrophils, decreased lymphocytes), consistent with effects on corticosterone concentrations (Pruett 2009). However, the atrazine

- induced decrease in natural killer cell activity was not mediated by changes in the HPA axis. The latter observation suggests other mechanisms besides HPA activation in atrazine

- induced decreases in natural killer cell activity. Pruett et al. (2009) provided some supporting evidence that 28

- day atrazine exposure did not cause habituation o r tolerance to the HPA axis perturbations, but immune function (e.g., antibody production) was not evaluated in this study.

It is important to note that the corticosterone response was stimulated by intraperitoneal injection of atrazine. Pruett et al. ( 2003) demonstrated that a single high oral dose of 500 mg/kg atrazine also increased endogenous corticosterone levels. This study was intended to provide insight into the predictive value of corticosterone production over time for impairments of the immun e system not to assess the immunotoxicity of atrazine per se

. While the available studies suggest a potential effect of atrazine on the immune system as noted in studies prior to 2003, conflicting results were reported in the two in vivo developmental imm unotoxicity studies available. Rooney et al. (2003) reported immunosuppression of cellular and humoral immune function in male Sprague

- Dawley rats, whereas Rowe et al. (2006) reported immunopotentiation of immune function in male Balb/c mice. The reasons for the contradictory results are not known but may be related to differences in the timing and route of maternal exposure, species differences and different methodologies used to assess immune response. Rooney et al. examined Sprague

- Dawley rats followi ng oral exposure

– a relevant route of human exposure

– from GD 10 until PND 23 to 35 mg/kg/day atrazine. Delayed type hypersensitivity response and the IgM antibody response to sheep red blood cell antigen were measured. In comparison, Rowe et al. used Balb/c mice which were exposed subcutaneously to atrazine for 21 days via time release pellets beginning between GD 10 and 12 through PND 10. The starting dose was 35 mg/kg/day beginning at GD10 and then decreased to approximately 23 mg/kg/day when the m ice gave birth, due to a constant rate of atrazine release from the implant that was not adjustable for increasing maternal body weight. T cell function (proliferation and cytolytic activity) after in vitro allogeneic stimulation was measured. Humoral re sponse was assessed after immunization with heat killed streptococcus pneumoniae . Finally, it should be noted that the results reported by Rooney et al. have not been reproduced by the same laboratory that conducted the first experiments.

It is not known if the developmental immunological effects of atrazine reported by Rowe et al. and Rooney et al. represent HPA axis alterations similar to those seen in adults at much higher doses. Rooney et al. (2003) provided some evidence that immune changes associated with atrazine during early development are not mediated throughPage 123 of 210 the suppression of prolactin o r thyroid hormones. The precise underlying mechanism of an atrazine

- mediated sex

- specific effect, however, is not fully understood.

In vitro studies intended to provide mechanistic data that would further clarify atrazine’s effect on the immune system, i ncluding intracellular signal transduction, have also been conducted. The underlying mechanisms of atrazine

- induced inflammatory effects such as nociception and plasma extravasation, may be mediated through mast cell degranulation and activation of the Gq /11

- protein coupled neurosteroid receptor, phospholipase C and intracellular Ca 2+ (Mizota and Ueda 2006). However, the inflammatory responses were examined following intraplantar administration of atrazine, a route of exposure that is not likely to occur in humans. Pinchuk et al. (2007) provided in vitro evidence suggesting that atrazine directly targets dendritic cell maturation and decreases major histocompatibility complex 1 (MHC

- 1) molecule expression on the dendritic cell surface.

Atrazine

- induce d inhibition of in vitro human peripheral blood lymphocyte NK cell

specific lysis of K562 human leukemia cells may be the result of inhibition of lytic granule release, without affecting the ability of the natural killer cell to form stable conjugates with target cells (Rowe et al. 2007). Using human peripheral blood mononuclear cells, the in vitro study of Devos et al. (2003) suggests that atrazine

induced inhibition of human peripheral blood mononuclear cell proliferation and cytokine production is most likely not mediated through nuclear receptors such as the glucocorticoid receptor, androgen receptor, members of the peroxisome proliferator

activated receptor family, and the retinoid

- related orphan receptor α.

Although in vitro studies provided some insight into potential mechanisms of atrazine

mediated immunotoxicity, the extrapolation of these mechanisms to the interpretation of immunotoxicity observed in vivo is difficult. Unlike the in vitro situation, in vivo the immune system’s response to atr azine exposure involves complicated feedback communication pathways with the central nervous system operative (for example, potential modulation of immune cell function by endocrine hormones such as corticosterone).

In summary, the recent studies sugges t a potential for atrazine to affect the immune system. However, the underlying modes of action of atrazine

- mediated immunotoxicity and its relevance to potential adverse health effects in humans are still not thoroughly understood. In adult rodents, the indications of immunotoxicity reported in the atrazine database may be due to direct interactions between atrazine and/or its metabolites and immune system cells. Alternatively, the immunotoxicity response could be related to endocrine hormones acting on immune cells. Consequently, no conclusions may be drawn at this time on the relationship between the immunotoxic effects observed after atrazine exposure and the activation of the HPA axis with the subsequent disruption of the HPG axis. Furthermore, dat a regarding the developmental immunotoxicity potential of atrazine is inconclusive since the two available developmental immunotoxicity studies yielded conflicting results. Nonetheless, the available data do not indicate atrazine

induced immunotoxicity is a more sensitive endpoint than the atrazine

- induced effectsPage 124 of 210 on neuroendocrine function, e.g., attenuation of LH surge in female Sprague

- Dawley rats which is used for the basis of the points of departure for the intermediate and chronic exposure scenarios

.

A.6.1 Cimino

- Reale, G., Ferrario, D., Casati, B., Brustio, R. Diodovich, C., Collotta, A., Vahter, M., Gribaldo, L. (2008). Combined in utero and juvenile exposure of mice and atrazine in drinking water modulates gene expression and clonogenicity of myeloid progenitors. Toxicol. Letters 180:59

- 66.

In this study, groups of four adult female CD

- 1 mice were exposed to 1mg/L sodium arsenate (NaHAsO 4 ) in drinking water for 10 days before mating and during gestation. Other groups of NaHAsO 4 - exposed m ale and female (4/sex/group) offspring were treated for 4 months with 1 mg/L atrazine (unreported purity), an additional dose of 1mg/L NaHAsO 4, or a combination of atrazine + NaHAsO 4 in drinking water. At the end of treatment, animals were sacrificed and bone marrow cells were collected from one femur, frozen and used for the colony forming unit

- granulocyte/macrophage (CFU

- GM) assay. Cells from the second femur were harvested, flushed with 1 mL of commercial RNA

- Later to stabilize the RNA for later isolat ion, and held frozen until use in the gene expression phase of testing.

For the CFU

- GM assay, bone marrow cells were thawed, centrifuged, and resuspended in culture medium specific for murine hematopoietic cells containing 30% fetal bovine serum, bovine pancreatic insulin, human transferrin iron

- saturated, 2

- mercaptoethanol, interleukin

- 3 and

- 6, and stem cell growth factor. Cell viability was determined; cells were adjusted to 6.0 x 10 5 viable cells/mL, seeded to yield a final density of 4.0 x 10 4 viable cells/plate and incubated. Triplicate plates were prepared per group and five replicated experiments were conducted. After 7 days, colonies were counted to determine cytotoxicity.

For the gene expression phase of testing, total RNA was extracted from bone marrow cells, and [ 33 P]

- labeled cDNA was obtained by converting 1 μg of total RNA into cDNA using reverse transcriptase in the presence of Mouse Cancer CDS primer mix. The cDNA was purified, hybridized for 16 hours on microarray membranes, washed, a nd exposed on a phosphor

- image screen for 21

- 24 hours. Transcripts were considered to be differentially expressed if the p value was ≤0.05.

Bone marrow cells were also investigated for modulation of gene expression of estrogen receptors alpha and beta u sing real

- time PCR. For this approach, 500 ng of total RNA, extracted from the bone marrow of treated males and females, was converted to cDNA through reverse transcription using random hexamer primers High

Capacity cDNA Archive Kit and Moloney murine leu kemia virus reverse transcriptase.

No gross pathological changes were seen in the treated mice. A significant (p<0.05) ~19% (males) and ~23% (females) decrease in the percentage of CFU

- GM was seePage 125 of 210 after exposure to 1 mg/L atrazine alone. NaHAsO 4 had no cytotoxic effect on the bone marrow. However, cell growth increased significantly (p<0.05) by 12% (138 vs. 123 colonies) after the combined atrazine/ NaHAsO 4 treatment of females, compared to control. Results from the microarray analysis showed that the re was no significant change in gene expression in bone marrow cells following exposure to either atrazine alone or NaHAsO 4 . However, the combined exposure to atrazine/NaHAsO 4 demonstrated the overexpression of 20 differentially expressed genes (DEGs). Fo r the analysis of changes in gene expression of estrogen receptors alpha and beta For the analysis of changes in gene expression of estrogen receptors alpha and beta (ERα and β), quantitative RT

- PCR of exposed bone marrow cells revealed that the ERα was no t modified in either sex after dosing with the three treatment groups. However, ERβ gene expression was significantly increased in both sexes and all treatment groups. Nevertheless, the high variability among individual females in expression of either ER α or β genes limits the interpretation of these data. CFU

- GM assay, quantitative RT

- PCR of exposed bone marrow cells revealed that the ERα was not modified in either sex after dosing with the three treatment groups. However, ERβ gene expression was signi ficantly increased in both sexes and all treatment groups.

Reviewer’s Comments: With the exception of the CFU

- GM assay results showing inhibition of myeloid cells, suggestive of an adverse effect on the immune system, no definitive conclusion can be reached that would shed light on the mechanism(s) responsible for the bone marrow immunotoxicity. This is because of the lack of an effect on gene expression in the treated bone marrow and the wide variation in the gene expression of ERβ among individual females.

A.6.2 Coder, P. S. (2010) An Oral (Gavage) Immuno toxicity and Hormone Evaluation Study for the Effects of Atrazine on Male Rats, WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH. Unpublished. (TK0023175)

In an oral gavage, 28

- day immunoxicity and hormone evaluation study, 71

- day old male Sp rague

- Dawley [Crl:CD(SD)] rats (10/subset/group) were administered Atrazine (ATR) (98.8%) in a 1% methylcellulose deionized water vehicle by oral gavage at 0, 6.5, 25, 100, and 0 pair

- fed control mg/kg. These doses were equivalent to 0, 1.3, 5, 20, and 0 mg/mL. Positive control animals were given either of the following: substance I

– Anti Asialo GM1 (rabbit) or substance II

– cyclophosphamide monohydrate and also used a pair

- fed control group (table attached, pg 29

- WIL 639072 study). The vehicle for the test and control substances was the same. Animals were examined twice daily for signs of mortality and morbidity. Individual clinical observations of toxicity were recorded 2 hours following dose administration. Food consumption and body weight were rec orded daily. Urine examinations were collected in certain subsets only to assess urinary corticosterone levels. Clinical, hematological and hormonal parameters were measured at termination. All animals were euthanized following dose administration of at razine (within 30 minutes) by decapitation. Hormonal analysis was performed on corticosterone, adrenocorticotrophic hormone (ACTH), progesterone, estradiol, estrone, estriol, and prolactin. Bioanalysis was done on androstenedione, testosterone, andPage 126 of 210 dihyd rotestosterone (DHT). Tissues were collected following blood sampling and organ weights were recorded for adrenal glands, intestinal tissue with Peyer’s patches, liver, spleen, thymus, lymph nodes (axillary, mandibular, mesenteric). All tissues were fixe d from subsets A

- D and tissues from subsets C & D were evaluated histopathologically. The spleen from animals assigned to subset D were used to perform the NKC Function assay. Those animals in subset E were used for the splenic AFC assay following immuni zation with sRBC on study day 24 (table attached, pg 29

- WIL 639072 study). On day 28, blood samples were taken and the harvested spleen tissues were used to determine the specific IgM antibody

- forming cells.

Data acquisition and reporting was done by W IL Laboratories, as was the statistical testing with the exception of daily body weight and food consumption data. The AFC and NKC functional assay data and organ (spleen/thymus) weight data were analyzed by ImmunoTox®, Inc.

Concentration analysis of g roup 2 (1.3 mg/mL.) describes the relative standard deviation (RSD%) created on March 22 (2.5%) as nearly two times that of the formulation created on April 12 (1.3%).

No treatment related mortality and morbidity were observed. Few atrazine

- related cli nical signs of toxicity were noted, with only the 100 mg/kg/day dose group displaying evidence of salivation after 2 hours following dose administration. There were no other test substance

- related clinical findings for animals at any dose level within 2 h ours of dosing, or during daily examinations. The positive control group II showed an increase of red, brown, and yellow fur staining, but no other remarkable clinical findings. No clinical signs were noted in doses at 25 or 6 mg/kg/day.

Treatment resul ted in dose

- dependent, statistically significant decreases in body weight gain and food consumption for ATR

- treated rats in the 25 and 100 mg/kg/day dose groups when compared to the untreated vehicle control group. The decrease found in body weight and fo od consumption persisted throughout the treatment period. Mean body weights for rats treated in the 6.5 mg/kg/day group were found to be ‘marginally’ statistically significantly lower than the vehicle control group during study days 10

- 24, only when those data from subsets D & E were combined. The analysis for subsets D and E were combined from day 1 to 24, because animals in these subsets were not necessarily comparable beyond day 24 (pg. 415

- WIL 639072 study). Food consumption for rats in this same dos e group did not show a consistent effect of treatment on subsets, while individually there was also no effect of treatment on food consumption in the 6.5 mg/kg/day group.

Hematology data was assessed in only subsets C & D. Rats treated for 14 days with ATR at any dose showed no test substance related alterations. In subset C, statistically significant variations from the vehicle control group were higher in mean corpuscular hemoglobin concentrations (MCHC) in the 25 an d 100 mg/kg/day groups, higher mean percent neutrophil count in the 100 mg/kg/day group, and a lower percent eosinophil count as well. These results were considered not toxicologically relevant since absolutePage 127 of 210 counts are more relevant for interpretative p urposes, hence these values did not show statistically significant or pathologically meaningful differences. Subset D, however, showed that treatment with ATR for 28

- days displayed multiple test substance related and statistically significant alterations in red blood cell parameters in the 100 mg/kg/day group when compared to the vehicle control and pair

- fed control group. These include low hemoglobin concentration and hematocrit, higher MCHC, reticulocyte counts (absolute and percentage), red cell distri bution width, and hemoglobin distribution width values. No other variations in hematology were attributed to test substance administration.

Subset C data displayed no cytological alterations or differences in bone marrow smear cytology between the contro l and the atrazine

- treated groups following 14 days of atrazine treatment. Overall ATR treatment at doses 25 and 100 mg/kg/day were associated with a rapid reduction in absolute thymus weights within 30 minutes following the first dose, although not found to be statistically significant. However statistical significance was found in 100 mg/kg/day group on days 0, 6, 13, and 27 when compared to the control group. There appeared to be no adverse toxicologically relevant effects of ATR treatment on thymus w eights at any dose and there were no ATR related effects revealed on any other organ weight.

Microscopic evaluation showed that animals euthanized after 14 days of treatment showed a 50% increase in periportal glycogen content in the liver and an increa sed absolute and relative liver weight in this group. There were no treatment effects evident in the 25 or 6.5 mg/kg/day groups. There appeared to be a trend towards a dose

dependent increase in Adrenocorticotrophic Hormone (ACTH) within 30 minutes follo wing ATR administration at all dose groups. However, statistical significance was not found due to a high degree of inter

- animal variability. No effect on ACTH was found after 7, 14, or 28 days of treatment. Dose

- related, statistical significance was fo und in plasma corticosterone levels in all ATR treatment groups on study day 0 but not on study day 6, 13, or 27. Urinary corticosterone levels were unaffected by ATR treatment on days 9 and 27. Plasma progesterone levels were statistically significantly elevated after the first dose on days 7, 14, and 28 at doses of 25 and 100 mg/kg/day in ATR treated groups. Prolactin plasma levels were highly variable and no noted effects of ATR treatment in any dose at any time interval. In plasma corticosterone and prolactin, the pair

- fed control group, on day 27 displayed an increase when compared to the vehicle control group.

Atrazine had no effect on male (androgens) or female (estrogens) sex hormones at any level at any interval. Aldosterone levels in ATR trea ted groups on study day 0 were higher than control groups on the same day, while statistical significance was found in the 100 mg/kg/day treated group. Any other changes found in other dose groups were considered not related to the ATR administration. Th is was based on changes in control levels with time, variability in the magnitude and the direction of the changes observed over time, and the absence of a dose

- response in the ATR treated groups.Page 128 of 210 Immunotoxicity assessments showed that ATR treatment at an y dose did not suppress the IgM AFC response when compared to controls, even at doses which caused statistically significant decreases on food intake and body weight gain over 28 days. Total spleen activity showed an enhanced AFC response in the 25 mg/kg/ day dose group only, although it was not considered biologically significant since no dose

response was observed and the increase was further attributed to a limited number of high responding animals. The positive control group I displayed a substantial a nd significant reduction in AFC activity.

Exposure to ATR at any dose didn’t decrease the ability of Natural Killer (NK) cells to kill target tumor cells. The 100 mg/kg/day dosed animals displayed an enhanced ability to kill target tumor cells compared t o vehicle control group, with a percent kill at effector to target ratio 200 to 1 and 100 to 1. Significantly suppressed NK cell activity was observed in the positive control group at all effector (NK cell) to target (Tumor cell) ratio. The pair

- fed contr ol group had higher responses than the vehicle controls, but none of the increases reached a level of statistical significance. There was no evidence that the pair

- fed animals showed any reduction in the cell mediated immunity AFC response or in NK cell a ctivity although a slight, but not statistically significant increase in NK cell activity was observed in the pair

- fed control group.

In conclusion, treatment of the male rats with ATR orally for 28 days did not ultimately produce adverse biologically rel evant effects on the AFC and NK assay, while other components of the immune system were not assessed for their function

– it is premature to definitively conclude that absolutely no functional deficits in humoral and innate immunity occurred. The study has shown equivocal evidence of toxicity to the immune system; further the immune system has not been proven to be a primary target organ. The toxicology database for atrazine does not indicate that the immune system is the primary target organ. The observe d effects on the immune system have been well characterized in the adult rat and these effects were seen at doses that produce overt systemic toxicity, i.e., decreased thymus weight at the highest does tested without histopathological findings.

The NOEL for immune suppression is found to be 25 mg/kg/day for this study. The LOEL was the HDT, or 100 mg/kg/day. This is based upon a decrease in thymus weight and NK cell activity at effector : target site.

A.6.3 Devos S, Bosscher K De, et al. 2003. Inhib ition of cytokine production by the herbicide atrazine

– Search for nuclear receptor targets. Biochemical Pharmacology 65: 303

– 308.

The purpose of this study was to test the hypothesis that atrazine affects leukocyte function via nuclear receptors suc h as the glucocorticoid receptor (GR), the androgen receptor (AR), members of the peroxisome proliferator

- activated receptor (PPAR) family, and the retinoid

- related orphan receptor (ROR) α. The authors noted previous work that showed weak binding of atraz ine to the AR. The GR was investigated because of the similarity of effects induced by atrazine and the synthetic glucocorticoidPage 129 of 210 DEX. Glucocorticoids have anti

- inflammatory and immunosuppressive properties and interfere with nuclear factor

– κB (NF

- κB ) and AP

- 1, and in this way repress the expression of inflammation

- related genes such as interleukin (IL)

- 6, IL

- 8, ICAM, and E

selectin, that contain NF

- κB and AP

- 1 response elements. Activation of PPARs has been reported to have effects on leukocytes. RORα can also affect leukocytes function.

In this study, adult (2

- or 7

- month

- old) CF1 mice were injected i.p. on days 1 and 4 with atrazine (100 mg/kg). Control mice received the vehicle only (carboxymethylcellulose containing 0.5% Tween 80). Mice were sacrificed on day 7. Liver and kidneys were removed. Brief descriptions of cells (murine fibrosacrcoma L929sA and rabbit kidney RK13) and culture procedures, plasmids (p1168hu.IL6P

- luc+, p98.IL8p

- luc+, p133.IL8p

luc+, pELAmP

- luc+), transfection and repo rter gene analysis, ELISA, AR assay, and peroxisome analysis were provided.

Inhibition of human peripheral blood mononuclear cell (PBMC) proliferation and cytotokine production was observed in cultures with atrazine (3 x 10

- 7 M to 1.5 X 10

- 5 M). This e ffect was not prevented by the glucocorticoid inhibitor RU486. In contrast to DEX, atrazine did not reduce IL

- 8 production. The authors concluded that these data suggest that the effects of atrazine on PBMC are not mediated through the GR. Atrazine did not affect TNF

- induced IL

- 8 promoter activity, in contrast to DEX. This was demonstrated in stable cell pools transfected with an IL

- 8 promoter

- driven reporter gene construct, p98.IL8p

- luc+, and then induced with TNF and treated with DEX and atrazine (3, 0.3, and 0.03 μM) alone or in combination. TNF is an inflammatory stimulus activating the reporter genes. Testing a longer variant of the IL

- 8 promoter, p133.IL8p

luc+, yielded the same overall results (data were not shown). To test whether the results with the IL

- 8 promoter were promoter

- specific, the authors also tested the effect of atrazine on other proinflammatory gene construct, containing the E

- selectin promoter (with NF

- κB elements) or the IL

- 6 promoter (with NF

- κB and AP

- 1 elements), that are both inducible by TNF. The authors indicated that there were no substantial effects with atrazine. They concluded that atrazine does not exhibit glucocorticoid

- like properties and does not interact with the GR.

No peroxisome proliferation was observed in the liver or kidneys of mice treated with atrazine. Moreover, no PPAR

- mediated induction of promoter activity was seen on targets of PPARα, PPARγ, or PPARδ. No binding of atrazine to the AR was observed. The a uthors concluded that their study shows that AR, GR, PPARs, or RORα are not likely targets of atrazine.

Reviewer’s Comments: Using human peripheral blood mononuclear cells, the in vitro study of Devos et al. (2003) suggests that atrazine

- induced in hibition of human peripheral blood mononuclear cell proliferation and cytokine production is most likely not mediated through nuclear receptors such as the glucocorticoid receptor, androgen receptor, members of the peroxisome proliferator

- activated recepto r family, and the retinoid

- related orphan receptor α. The relevance of this study to human health risk assessment is limited since it entails intraperitoneal injection of atrazine to the animals prior to in vitro experiments.Page 130 of 210 A.6.4 Devos S, Van Den Heuve l R, et al. 2004. Limited effect of selected organic pollutants on cytokine production by peripheral blood leukocytes. Eur. Cytokine Netw: 15: 145

– 151.

This study examined the potential effects of in vitro atrazine treatment on cytokine productio n by peripheral blood mononuclear cells (PBMC) from control and allergic donors. Cells were cultured for six days in the presence of atrazine (3 μM) or vehicle with phytohemagglutinin (PHA) or Dermatophagoides pteronyssinus (Dpt) extract, then for one day in the presence of PHA + phorbol 12

- myristate 13

- acetate. Information on the donors (selection, ethical approval, allergic disease description) and methods (PBMC cultures, cytokine assays, IgE plasma level determination, radioallergosorbent or RAST test to confirm Dpt allergy) was provided.

Atrazine treatment significantly reduced cytokine production IL

- 5 and IFN

- γ in the donors. This effect was apparent in PHA

- stimulated cultures, but not Dpt stimulation.

Reviewer’s Comments: The authors concluded that since the effects on cells from allergic and non

- allergic donors were similar, and the effects on cytokines were limited, the impact of atrazine on allergic responses is unlikely.

A.6.5 Filipov, N M, Pinchuk, L M, et. al. 2005. Immunotoxic effects of short

- term atrazine exposure in young male C57BL/6 mice. Toxicological Sciences: 86(2): 324

- 332.

The objective of this study was to assess the effects of atrazine exposure on selected immune parameters in young mice. Parameters included: thymus an d spleen weights, cellularity, lymphocyte subpopulations in the spleen, thymus, and peripheral blood mononuclear cells. To assess short

- vs. long

- term effects, the parameters were measured at one day, one week, and seven weeks after administration of the last atrazine dose. Atrazine was administered to one

- month

- old male C57BL/6 mice for 14 days by oral gavage at doses of 5, 25, 125, and 250 mg/kg bw.

Spleen weight was decreased at the 250 mg/kg dose level on day 1, while thymus weights were decreased at the 125 and 250 mg/kg/day dose levels. Decreased thymus weights persisted one week after the last dose of atrazine, but resolved by seven weeks. Spleen and thymus cellularity was dose

- dependently decreased by atrazine treatment (statistically signif icant at 125 and 250 mg/kg doses) at day 1. The decreased cellularity persisted one week after cessation of atrazine treatment. However, decreased thymic cellularity was resolved by seven weeks, whereas decreased splenic cellularity was significantly d ecreased at all doses levels of atrazine. Thymic T

- cell populations were significantly and dose

- dependently decreased at day 1. The authors reported that one week after cessation of atrazine treatment, the decreases in most T

- cell populations were still present at the higher doses of atrazine, whereas at the lower doses a non

- significant rebounding effect was observed (data not shown). The authors also reported that by seven weeks after the last atrazine dose, thePage 131 of 210 decreases in the thymic subpopulations w ere numerically present, but were no longer significant (data not shown).

In regard to splenic cell phenotypes, both MHC II+ and CD19+ cells were dose

dependently decreased, whereas only the high dose of atrazine increased the percentage of CD3+ cells. In addition, atrazine treatment decreased the number of splenic naïve T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. The proportion of mature splenic dendritic cells (DC; CDIIc high ) was also decreased, which persisted for at least one week. No effects on splenic cell phenotypes were seen at seven weeks post treatment time point.

With respect to peripheral blood mononuclear cells, a dose

- dependent decrease in circulating CD4+ cells w as observed one day after the last atrazine dose. The decrease in CD4+ cells was still present at the 250 mg/kg dose (high dose) at seven days after the last dose. In addition, at seven days, the MHC

- II+ cells were also decreased at the high dose. The a uthors stated that most of the changes in peripheral blood mononuclear cells were no longer apparent at seven weeks after the last atrazine dose, although data were not shown. The authors concluded that “atrazine exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.”

Reviewer’s Comments: Although T cell subpopulations were decreased at all dose leve ls on Day 1 the fact that they quickly recover diminishes the toxicological significance of this finding ( i.e., its ability to predict impaired function). A similar conclusion was reached by The Joint FAO/WHO Meeting on Pesticides Monograph on Atrazine (JM PR 2007).

A.6.6 Karrow, N A, McCay, J A, et. al. 2005. Oral exposure to atrazine modulates cell

- mediated immune function and decreases host resistance to the B16F10 tumor model in female B6C3F1 mice. Toxicology: 15

- 28.

The purpose of this study was t o examine the potential immunomodulatory properties of atrazine and was designed to assess changes in cellular and antibody immune function in the female B6C3F1 mouse model. This model also included pathogenic B16F10 tumor and Listeria monocytogenes chall enge models to assess potential biological significance of any observed changes in immune function. In this study, female B6C3F1 were administered atrazine by gavage (0, 25, 250, and 500 mg/kg/day) for 14 days then evaluated for immune responsiveness. Im mune assays and toxicity parameters included: body weights, organ weights (including spleen), hematology, differential cell counts, serum IgM titers, splenic natural killer (NK) cell activity, splenic mixed lymphocyte response (MLR), splenic IgM antibody

- forming cell (AFC) response, reticulocyte counts, splenic lymphocyte proliferation, splenic B and T cell enumeration, splenic cytotoxic T lymphocyte response (CTLR) to P815 mastocytoma cells,Page 132 of 210 mononuclear phagocytic system (MPS) evaluation, L. monocytogenes challenge and B16F10 tumor challenge.

A decrease in body weights was observed in the atrazine treated groups. A significant dose

- dependent decrease in spleen and thymus weights was also observed. Kidney weights were slightly but significantly reduced at the high dose. Hematological alterations included reductions in erythrocyte counts (all dose levels), decreased hematocrit (high dose), and increased mean corpuscular hemoglobin (all doses). At the high dose, the percentage of CD3 + and CD4 - CD8 + T ce lls was significantly increased by 10 and 18%, respectively. Atrazine treatment did not significantly alter the spleen cell proliferative response to mitogens Con A or LPS. There was no effect of treatment on splenic primary AFC response and serum IgM ti ters to sRBCs. The spleen cell MLR to DBA/2 murine spleen cells was stimulated (30%) in the high

- dose mice. The splenic CTLR was significantly stimulated in mice that were treated with both 25 and 250 mg/kg atrazine. The NK cell activity in the spleen was only inhibited in the 250 mg/kg/day group and the authors did not consider this effect to be of biological significance. A dose

- related decreasing trend in MPS function across the treatment groups was noted.

Host resistance to B16F10 tumor challeng e was reduced in atrazine treated mice at both challenge levels. At the 3 x 10 5 cells/mouse challenge level, a significant dose

dependent increase in the number of B16F10 nodules was observed in the lungs of treated mice. The greatest increase in the num ber of nodules (122%) was observed at the 500 mg/kg treatment level. Although there were no statistically significant changes in host resistance to L. monocytogenes, the authors stated that the data suggest that atrazine may alter host resistance to this pathogen. The percent mortalities for the various treatment groups at the challenge level of 6.3 x 10 4 CFU/mouse were 67, 92, 58, and 33 for the control, 25, 250, and 500 mg/kg atrazine treatment groups.

Reviewer’s Comments: This study design included a host resistance test, which assessed the function of the immune system in toto.

A.6.7 Mizota K and Ueda H. 2006. Endocrine disrupting chemical atrazine causes degranulation through Gq/11 protein

- coupled neurosteroid receptor in mast cells. Toxicologi cal Sciences 90(2): 362

– 368.

The authors examined the in vitro effects of several putative endocrine disrupting chemicals including atrazine on β

- hexosaminidase release from mast cells and their putative neurosteroid receptor involvement. Drawing from their previous research, the authors proposed that NS2

- type neurosteroid receptors exist on mast cells and mediate histamine release, which, in turn, stimulates nociceptor endings. They previously demonstrated that neurosteroids cause extravasation as we ll as hyperalgesia, which were abolished by diphenhydramine, a representative H1 receptor antagonist. Neurosteroid

- induced extravasation has been attributed to degranulation of mast cells.Page 133 of 210 Degranulation of mast cells was determined by measuring the rel ease of granule

associated β

- hexosaminidase from RBL

- 2H3 cells. Degranulation was also evaluated by Alexa 488

- annexin V binding to the phosphatidylserine, which is externalized during degranulation. Antagonism of atrazine

- induced Alexa 488

- annexin V bind ing in RBL

2H3 cells by BSA

- conjugated progesterone (PROG

- BSA) was examined. β

Hexosaminidase release was characterized by various inhibitors including antisense

oligodeoxynucleotide for Gαq/11, pertussis toxin, phospholipase C inhibitor U

- 73122, inosito l 1,4,5

- triphosphte receptor inhibitor xestospongin C and Ca2+ channel blocker lanthanum chloride.

Based on the authors previous findings that intraplantarly administered neurosteroids cause pain

- like responses, such as biting and licking behaviors in a diphenhydramine

reversible manner, these responses were also evaluated in male ddY mice. Atrazine (10 fmol) was administered intraplantarly (i. pl.) into the hind paw. The total amount of time showing pain responses, such as biting and licking, was meas ured for 10 min. after i.pl. injection.

Atrazine (10 nM

– 1 μM) caused a concentration

- dependent degranulation in RBL

- 2H3 cells and peritoneal mast cells as measured by β

- hexosaminidase release. This release was blocked by PROG

- BSA, a membrane impermea ble neurosteroid receptor antagonist. PROG

- BSA also blocked atrazine

- mediated increase in Alexa 488

- annexin V binding (which labels phosphatidylserine externalization in the plasma membrane representing degranulation) in RBL

- 2H3 cells. The authors indic ated that the observation of atrazine

- induced increase in Alexa 488

- annexin V binding shows that the atrazine

- induced degranulation is unlikely due to cell damage.

Antisense

- oligodeoxynucleotide pre

- treatment reduced atrazine

- induced β

hexosaminidase re lease. Atrazine

- induced degranulation was abolished by phospholipase C inhibitor U

- 73122. Lanthanum chloride, which inhibits Ca2+ influx in cells, had no effect. The authors interpreted the findings to reveal that the signal transduction involved in th e degranulation is mediated though G q/11 , PLC and mobilization of intracellular Ca2+, characteristics of neurosteroids.

The results also demonstrated atrazine

- induced pain response that occurred immediately and ceased within 5 minutes after i.pl. administration. The responses were dose

- dependent between 1 and 10 fmol (i.pl.). The effects were abolished by PROG

– BSA and DPH, which is a histamine receptor antagonist. This was interpreted by the authors to indicate that the responses were caused by neurosteroid receptor

- mediated histamine release from mast cells, which was supported by atrazine

- induced plasma extravasation assay using Evans blue. Overall, the authors concluded that atrazine induces some inflammatory effects, including nociception a nd plasma extravasation, and the underlying mechanisms are mediated through mast cell degranulation and activation of the G q/11 - protein coupled neurosteroid receptor, phospholipase C and intracellular Ca2+ mobilization.Page 134 of 210 Reviewer’s Comments: This study concluded that the underlying mechanisms of atrazine

- induced inflammatory effects such as nociception and plasma extravasation, may be mediated through mast cell degranulation and activation of the Gq/11

- protein coupled neurosteroid receptor, phospholipase C and intracellular Ca 2+ . However, the inflammatory responses were examined following intraplantar administration of atrazine, a route of exposure that is not likely to occur in humans.

A.6.8 Pinchuk L M, Lee S

- R, and Filipov N M. 2007. In vitro atra zine exposure affects the phenotypic and functional maturation of dendritic cells. Toxicology and Applied Pharmacology 223: 206

– 217.

Based on prior results revealing that atrazine may affect dendritic cell (DC) function (Rowe et al. 2006; Filipov et a l. 2005), the objectives of this study was to investigate the effects of in vitro atrazine exposure on the phenotypic and functional maturation of a growth factor

- dependent, bone

- marrow

- derived immature murine DC cell line JAWSII. For comparative studies , limited studies were performed with primary thymic DC. Details were provided on the following materials and methods used for the analyses: dendritic cell line, primary thymic DC preparation, antibodies and flow cytometry, endocytosis assay, apoptosis a ssay, PCR analysis, and real

- time PCR analysis.

JAWSII cells were induced to mature in the presence of growth factors and selected maturational stimuli in vitro . The DC line was exposed to atrazine (1

– 300 μM) and the effects on phenotypic and functio nal maturation of DC were examined. Phenotypic changes caused by atrazine included a dose

- dependent removal of surface MHC

- 1 with a significant decrease at 1 μM concentration. In addition, atrazine treatment decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. The phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation at higher conce ntrations of atrazine where receptor

mediated antigen uptake was increased. The authors stated that their data suggest that atrazine directly targets DC maturation and removes MHC

- 1 molecules from the DC surface.

Reviewer’s Comments: Pinchuk et al. (2007) provided in vitro evidence suggesting that atrazine directly targets dendritic cell maturation and decreases major histocompatibility complex 1 (MHC

- 1) molecule expression on the dendritic cell surface.

A.6.9 Pruett, S B, Fan, R, et al. 2003. Modeling and predicting immunological effects of chemical stressors: characterization of a quantitative biomarker for immunological changes caused by atrazine and ethanol. Toxicological Sciences: 75: 343

- 354.

The authors stated that the study was con ducted to determine if the “stress

- induced immunological effects of atrazine and ethanol could be predicted, and if it is feasible to use one immunological parameter as a biomarker of stress to predict the quantity ofPage 135 of 210 changes expected in other immunologica l parameters.” Consequently, doses were selected in order to elicit an increase in corticosterone levels as a predictor of immune function; this study was not intended as a hazard identification study of atrazine. Female C57BL/6 X C3H F1 (B6C3F1) mice (8

– 12 weeks of age following a two

- week accommodation period) were used in the study. Atrazine was administered intraperitoneally (only once) at dose levels of 100

- , 200

- , or 300

- mg/kg bw in corn oil (group size was 5 mice). A naïve control group was in cluded

. The authors selected corticosterone as a mediator that might predict the quantity of immunosuppression caused by stress responses. Therefore, the corticosterone area under the curve (AUC) values for atrazine

- treated mice were determined. Immuno logical parameters included: thymus and spleen cellularity, lymphocyte subpopulations in the thymus and spleen, expression of MHC class II protein on splenocytes, antibody responses to keyhole limpet hemocyanin, and natural killer cell activity. Models w ere derived describing the relationship between corticosterone AUC and potential immunological changes.

The peak corticosterone response (approximately 1200 ng/ml) to atrazine occurred at 6 hours, and was dose

- dependent. In order to assess if the observ ed corticosterone response was unique to the intraperitoneal route of exposure, the authors included an additional experiment in which five mice were treated by oral gavage (corn oil) to 500 mg/kg atrazine, while five mice were administered the vehicle. The 500 mg/kg dose was selected in order to match the high dose used in the National Toxicology Program’s study on atrazine. Three hours after dosing, serum corticosterone levels were measured. The authors reported the following corticosterone values: c ontrols: 164 ± 88; atrazine

- treatment: 676 ± 79.

Linear regression models of corticosterone AUC vs. thymus

- and spleen

- cell numbers and subpopulations revealed that atrazine treatment was associated with decreased thymus cell number and percentage of thymic CD4+CD8+, and decreased spleen total cell number and splenic B220+ cells. Linear regression models of corticosterone AUC vs. expression of MHC Class II protein on splenocytes indicated a dose

- dependent decrease in the expression of MHC Class I I protein on splenocytes in atrazine

- treated animals. The results of linear models also indicated dose

- dependent decreases in splenic NK cell activity and IgG1 and IgG2a responses to KLH.

The authors also assessed the potential correlations between obs erved effects of atrazine and effects predicted using models derived from mice treated with exogenous corticosterone or restraint stress with linear modeling. The authors stated that the modeling results indicated that the atrazine effects were similar to results obtained from mice subjected to restraint stress; however, some of the atrazine effects were greater than predicted on the basis of the stress response alone.

Reviewer’s Comments: Pruett et al. (2003) demonstrated that a single high oral dose o f 500 mg/kg atrazine also increased endogenous corticosterone levels. This study was intended to provide insight into the predictive value of corticosterone production over time for impairments of the immune system not to assess the immunotoxicity of atra zine per se

.Page 136 of 210 A.6.10 Pruett, S B, Fan, R, et al. 2009. Patterns of immunotoxicity associated with chronic as compared with acute exposure to chemical or physical stressors and their relevance with regard to the role of stress and with regard to immun otoxicity testing. Toxicological Sciences: 109(2): 265

- 275.

This study is an extension of an earlier study (Pruett et al. 2003) that entailed single dose exposure to atrazine. The purpose was to determine if mice can become tolerant or habituated wit h regard to the stress response and accompanying immunological effects.” Female C57BL/6 X C3H F1 (B6C3F1) mice (8

– 12 weeks of age following a two

- week accommodation period) were used in the study. Mice were dosed daily (intraperitoneally in corn oil) f or 28 days with atrazine (up to 150 mg/kg/day) or were subjected to restraint, which can also induce neuroendocrine stress responses and alter immunological parameters. The exact doses of atrazine were not specified except for the high dose of 150 mg/kg/d ay. At the end of the study, the following parameters were measured: white blood cell count and differential blood count, lymphocyte phenotypes in the blood and spleen, natural killer (NK) cell activity in splenocytes, and corticosterone concentrations ( at various times after dosing or restraint). The results were compared with those from the acute study (Pruett et al. 2003).

The authors provided results for the corticosterone response in mice treated with 150 mg/kg/day atrazine with that observed in the acute study (at 200 mg/kg dose level). The corticosterone response was very similar after the 28

- day treatment as observed after a single treatment. The authors concluded that the 28

- day atrazine treatment did not cause tolerance or habituation.

Restraint had little effect on white blood cell counts following one treatment or after a 28 day treatment period. Atrazine suppressed white blood cell counts; the suppression was greater after 28 day treatment compared with the single dose treatment. A trazine caused essentially the same increase in neutrophils and decrease in lymphocytes after 28 days of treatment as it did after 1 dose administration. Changes in the percentage of MHC class II expression by B lymphocytes and MHC class II expression on B lymphocytes in the spleen were comparable between the single dose and 28

- day daily dose regimens. Atrazine caused a similar decrease in B cell percentage in the blood and spleen in the 28

- day and single day studies. Restraint did not affect the perce ntage of B lymphocytes following acute or 28

- day treatment. A dose

- dependent decrease in CD4+ and CD8+ T lymphocytes in the blood was noted after single dose administration of atrazine; however, 28

- day treatment increased this parameter. Restraint for 2 8

- days decreased the T cell percentage in the blood, whereas acute restraint caused a smaller decrease. Decreases in NK cell activity of splenic lymphocytes were noted with both acute and 28

- day atrazine treatments; however, decreases were greater with th e latter treatment.

Reviewer’s Comments: Pruett et al. (2009) provided some supporting evidence that 28

- day atrazine exposure did not cause habituation or tolerance to the HPA axisPage 137 of 210 perturbations, but immune function (e.g., antibody production) was not e valuated in this study.

A.6.11 Rajkovic, V, Matavulj, M and Johansson, O. (2010). Combined Exposure of Peripubertal Male Rats to the Endocrine

- Disrupting Compound Atrazine and Power

- Frequency Electromagnetic Fields Causes Degranulation of Cutaneous Ma st Cells: A New Toxic Environmental Hazard? Arch. Environ. Contam. Toxicol. DOI 10:1007/s00244

- 010

- 9477

- 6

This study evaluated the in vitro effects of single and combined treatments of atrazine (98% purity) and power

- frequency electromagnetic fields (EMF s) on cutaneous mast cells in juvenile/peripubertal male Wistar rats. The investigators chose to study the effects of atrazine and EMFs because they thought it likely that humans could be exposed simultaneously to various combinations of chemical and phys ical environmental agents, which raises a concern for possible health effects. Atrazine was chosen since it is represents a chemical found in drinking water and EMFs generated by power lines and electric appliances represent an unavoidable physical factor in the environment.

Male Wistar rats were treated from postnatal day (PND) 23 to PND 53. Animals were divided into 6 groups (n=10 animals/group): 1) control ; 2) 4 hours/day to EMFs (50 Hz, 100

- 300 μT, 54

- 160 V/m); 3) 20 mg/kg/day atrazine ( per os , dissolved in olive oil); 4) 200 mg/kg/day atrazine ( per os , dissolved in olive oil); 5) combined EMF with 20 mg/kg/day atrazine; 6) combined EMF with 200 mg/kg/day atrazine. The exposure system for EMF treatments was previously described in Rajkovic et al . 2005a 15 . Dose levels were selected based on previous studies on atrazine effects on prepubertal rats (Stoker et al. 2000b 16 ).

Light Microscopy: Animals were decapitated and skin samples (10/group) were taken from the interscapular region, fixed in 4% pa raformaldehyde, embedded in paraffin, and sectioned into 5

- μm

- thick sections. The histochemical staining method with toluidine blue was used for the histological analysis and quantitative estimation of mast cells.

Stereological Analysis: Skin sections fro m 10 rats in each experimental group were analyzed stereologically using a multipurpose stereological grid M

- 42 placed in the ocular of a Reichert light microscope under a total magnification of x400. The numerical (Nvm) and volume (Vvm) densities of the total mast cell population of degranulated cells and of intact cells in the skin dermis were determined. Estimations were made by the same observer on blindcoded sections.

15 Rajkovic, V et al. 2005a. The effect of extremely low

- frequency electromagnetic fields on skin and thyroid amine

- and peptide

- containing cells in rats. An immunohistochemical and morphometrical study. Environ Res 99(3):369

- 377. 16 Stoker TE et al. 2000b. Endocrine

- disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid function in the male rat. A focus on the EDSTAC recommendations. Crit Rev Toxicol. 30(2):197

- 252Page 138 of 210 A.6.12 Rooney, A A, Matulka R, and Luebke, R. 2003. Developmental atrazine expo sure suppresses immune function in male, but not female Sprague

- Dawley rats. Toxicological Sciences 76: 366

- 375.

This study was designed to test the hypothesis that atrazine may cause developmental immunotoxicity through its disruption of prolactin (PRL ) or thyroid hormones during a critical early period of development. Pregnant Sprague

- Dawley (SD) rats were exposed via oral gavage to 35

- mg atrazine/kg/day from gestational days (GD) 10 (to avoid the possible interference of atrazine with embryo implanta tion) through postnatal day (PND) 23. The authors indicated that the dose of 35 mg/kg/day was set “just above the NOEL for atrazine

- induced developmental endocrine dysfunction to examine the potential for atrazine to indirectly induce immunotoxicity.” Th e authors stated that this dose is likely to induce endocrine effects in both sexes. Separate groups were exposed to bromocryptine (BCR) at 0.2 mg/kg/2x/day to induce hypoprolactinemia or to propylthiouracil (PTU) at 2 mg/kg/day to induce hypothyroidism. At 7 weeks old, the following immune functions in the offspring were evaluated: natural killer (NK) cell function, delayed

- type hypersensitivity (DTH) responses to bovine serum albumin (BSA), phagocytic activity of peritoneal macrophages, and antibody re sponse to sheep erythrocytes (SRBC).

There was an increase in offspring mortality in atrazine treated animals during PND 2

– 14. The average % offspring mortality/litter in atrazine

- treated dams was 13.2 compared to 2.3% for control dams. The authors noted that increased mortality was “unexpected, as this has not been reported for this dose of atrazine.” They further state that “it is not known whether the weight loss or death of pups was due to an effect on the physiology or behavior of the dams or the pups themselves.” The authors stated that male offspring body weight of the atrazine group was decreased relative to controls (mean control body weight was 20.1 g compared to a mean atrazine group body weight of 19.0 g).

The primary (IgM) antibody response to SRBC was suppressed in atrazine

- treated males (p = 0.0149), but not female, offspring at 8 weeks of age. There was no difference among treatment groups in the IgG antibody response to SRBC. There was no difference between the offspring of ATR

- exposed dams and the control offspring in the IgM antibody response to SRBC in 6

- month

- old

- animals (data were not shown). The latter results indicate that that immune function returned to normal by 6 months. The DTH response of male, but not female, off spring of atrazine treated dams was reduced relative to the control animals (p = 0.0308) when the animals were sensitized to BSA at 8 weeks of age and measured at 9 weeks of age. The decrease in DTH response of male offspring of the atrazine treatment gro up persisted until at least 12 weeks of age. There was no difference in the DTH response between the offspring of atrazine

- treated dams and the control offspring in 6

- month

- old animals (data were not shown). There was no effect of atrazine treatment on NK cell activity. There was no effect of atrazine treatment on phagocytosis by peritoneal macrophages.Page 139 of 210 The authors concluded that atrazine treatment causes gender

- dependent immunosuppression of cellular and humoral immune function, specifically suppres sion of primary antibody production and DTH responses. The authors also concluded that the immune changes associated with atrazine are not mediated through the suppression of PRL or THs. In addition, the authors concluded that “the current data do not su pport nor completely refute a relationship between atrazine’s immunotoxic potential and its endocrine disrupting properties.”

Reviewer’s Comments: Rooney et al. (2003) provided some evidence that immune changes associated with atrazine during early develo pment are not mediated through the suppression of prolactin or thyroid hormones. The precise underlying mechanism of an atrazine

- mediated sex

- specific effect, however, is not fully understood.

A.6.13 Rowe, A M, Brundage, K M, Schafer, R, and Barnett J B . 2006. Immunomodulatory effects of maternal atrazine exposure on male Balb/c mice. Toxicology and applied Pharmacology 21: 69

– 77.

The aim of this study was to test the hypothesis that prenatal/lactational exposure to atrazine would affect the deve lopment of the immune system and alter immune function in the adult offspring in the Balb/c mouse model. Pregnant Balb/c dams were exposed subcutaneously for 21 days via time release pellets to 700 ug atrazine/day beginning between days 10 and 12 of pregn ancy. The implanted pellet was designed to release the same amount of atrazine every minute of every hour of every day until the end of the treatment period. The rate of exposure at the beginning of treatment was reported to be approximately 35 mg/kg/day , however, the daily dose dropped to approximately 23 mg/kg/day by the time the mice gave birth. The authors reported that the prenatal/lactational exposure “caused no overt physical malformations in the offspring and had no effect on the number of litter s carried to term or the litter size.”

Immune status was evaluated at approximately 3 months of age. Body weight and spleen weights (absolute and relative) were monitored. T cell function was assessed by measuring proliferation and cytolytic activity after in vitro allogeneic stimulation. Allogeneic stimulation was assessed using two different assays. In one assay, splenocytes were isolated from unimmunized prenatally/lactationally exposed Balb/c mice and stimulated using irradiated splenocytes from female C57B1/6 mice. Proliferation of alloreactively stimulated lymphocytes was assessed by measuring the incorporation of 3 H

- thymidine into newly synthesized DNA following co

- culture with alloreactive stimulator cells and incubation with 3 H

- thymidine. In another assay, the ability of the T cells to lyse alloreactive target cells ( i.e. , cytotoxic T lymphocyte response), was assessed following alloreactive stimulation. EL

- 4 lymphoma cells were used as targets. The humoral immune response was assessed a fter immunization with heat killed Streptococcus pneumoniae (HKSP). The number of HKSP

- specific B cells was quantified by the ELISpot assay. Spleen cell phenotype (specifically, CD4 + , CD8 +

, and B220 + ), was determined by flow cytometry.Page 140 of 210 There were no changes in body weights or spleen weights. No changes were observed in the number of CD4 + T cell, CD8 + T cell, and B220 + B cell subpopulations in the spleen. Male mice (3

- month old) exhibited an increase in both T cell proliferation (atrazine offspring: 67 ± 5 x 10 3 cpm; control offspring: 46 ± 4 x 10 3 cpm) and cytolytic activity (approximately 66% increase). A signific ant increase in the number of HKSP

specific IgM secreting B cells in the spleen of treated male mice was noted. Male atrazine

- exposed offspring had 78.7 ± 4.5 HKSP

- specific IgM antibody secreting cells (ASC) per 10 6 B220 + cells compared to 58.6 ± 3.0 ASC per 10 6 B220 + cells for male control offspring. Three

- month

- old female offspring showed no significant difference in immune response from controls. The authors concluded that the results indicate “a male

- specific immunopotentiation of the immune response following prenatal/lactational atrazine exposure.”

In a review article (Rowe et al. 2008; Basic & Clinical Pharmacology & Toxicology, 102, 139

- 145), the study authors reported on the results of similar assays performed on available littermates at 6 m onths of age (defined as “long

- term”). Limited data were presented. The authors noted that too few male offspring were available to perform replicate assessments. However, they stated that a single iteration of the mixed lymphocyte response assay in the males showed a significant decrease (this contrasts with observations at 3 months of age). Female offspring were immunized with the HKSP vaccine at 6 months and sera collected at 7 and 14 days after immunization. The authors reported that six

- month

- old atrazine

- treated females had a significant and reproducible reduction in the number of phosphorylcholine (PC)

- specific responding splenocytes following HKSP vaccination (PC

- specific IgM

- producing B cells in the spleen of HKSP

- vaccinated atrazine

- treated f emales were 69% of control). In addition, spleens from the atrazine

- treated groups produced a mean of 72% of control IgG PC

specific B cells per million splenocytes. The MLR response was also assessed in female offspring. Results indicated that atrazine

- treated females had a significant decrease in the MLR (spleen cell proliferation level was 72% of control and was statistically significant).

The authors also stated that in the 6

- month

- old females, the total number of splenocytes and the lymphocyte subpopulation phenotypes were also unaffected. The mean spleen weight and total spleen cell number were slightly higher in the treated groups. The percentage of spleen cell subtypes (CD4, CD8, and B220) was not significantly different between the two gr oups. Based on the findings of the 3

- and 6

- month evaluations, the study authors concluded that the findings “demonstrate a sex

- and age

- dependent effect of prenatal exposure to atrazine on the immune system of the adult offspring.”

Reviewer’s Comments: Rowe et al. (2006) reported immunopotentiation of immune function in male Balb/c mice. However, these results conflicted with the results by Rooney et al. (2003) who reported immunosuppression of cellular and humoral immune function in male Sprague

- Dawley rats. The reasons for the contradictory results are not known but may be related to differences in the timing and route of maternal exposure, species differences and different methodologies used to assess immune response.Page 141 of 210 A.6.14 Rowe A M, Brundage K M a nd Barnett J B. 2007. In vitro atrazine

exposure inhibits human natural killer cell lytic granule release. Toxicology and Applied Pharmacology 221: 179

– 188.

Earlier studies have revealed effects of atrazine on natural killer (NK) cells. In the

curr ent in vitro study, Rowe et al. were interested in testing the hypothesis that atrazine exposure would decrease target cell adhesion and subsequent granule release in order to clarify precise changes in NK cell lytic function. Peripheral blood lymphocytes were isolated from healthy human male donors and exposed to atrazine (0, 10 or 30 μM) for 24 hours. The ability of NK cells to bind target cells (K562 human leukemia cells that are sensitive to lysis by human NK cells) as well as the ability of the NK ce lls to subsequently release lytic granule was assessed. Details on the methods used were provided in the study including: cell culture, peripheral blood lymphocyte harvest, cytotoxic lymphocyte assay, enrichment of NK cells, flow cytometry, NK cell adhes ion, perforin ELISA and western blots.

Results showed that atrazine decreased NK cell function of PBLs under the in vitro experimental conditions, confirming observations by other investigators, e.g., Whalen et al. 2003. The authors also reported that at razine did not affect viability or NK cell subpopulation distributions (data were not shown). For this experiment, PBLs were assessed for viability and subpopulation distribution via flow cytometry. Subpopulation distribution was determined using four ly mphocyte markers (CD56, CD16, CD3 and CD8), and viability was determined using 7

- ADD exclusion.

The basal level of lytic proteins ( i.e. , perforin, granzyme A and B levels) was not changed following a 24

- hour exposure to atrazine. Atrazine also did not affect the post

translational modification of perforin. In addition, atrazine treatment did not affect the ability of NK cells to bind target cells. However, atrazine treatment decreased the ability of NK cells to release granule proteins.

Reviewer’s Comments: Rowe et al. 2007 showed that atrazine

- induced inhibition of in vitro human peripheral blood lymphocyte NK cell

- specific lysis of K562 human leukemia cells may be the result of inhibition of lytic granule release, without affecting the ability of the natural killer cell to form stable conjugates with target cells.

A.6.15 Schwab, C L, Fan R, et al. 2005. Modeling and predicting stress

induced immunosuppression in mice using blood parameters. Toxicological Sciences 83: 101

– 113.

This study is an extension of the Pruett et al. (2003) study described above except that the blood rather than the spleen was of interest. Utilizing the same approach as Pruett et al. (2003), Schwab et al. (2005) showed that the atrazine

- induced decreases in CD4+

a nd CD8+ T cells in blood were similar to exogenous corticosterone.

Reviewer’s Comments: This study provides further support for the predictive value of corticosterone for immune markers.Page 142 of 210 A.6.16 Whalen M M, Loganathan B G, et al. 2003. Immunomodul ation of human natural killer cell cytotoxic function by triazine and carbamate pesticides. Chemico

- Biological Interactions 145: 311

– 319.

The effects of in vitro atrazine treatment on the ability of human natural killer (NK) cells to lyse tumor cells were investigated. Atrazine was tested in both purified NK cells as well as a cell preparation that contained both T and NK lymphocytes (T/NK cells). Lymphocytes were exposed to atrazine (10 μM) for periods of time ranging from 1 hour to 6 days. Inform ation on the methods used (isolation of NK cells, cell treatments and cell viability, and chromium release assay for NK cytotoxicity) was presented in the study.

Exposure of highly purified NK cells to atrazine inhibited K562 tumor cell lysis by 63 ± 25 % after 24

- hour exposure, and by 83 ± 21 % after a 6

- day exposure. Atrazine also caused significant decreases in lytic function when T/NK cells were exposed for 24

- hours (61 ± 13%) and after 6

- days (54 ± 12%).

Reviewer’s Comments: This in vitro study p rovides some evidence that atrazine may affect NK cell cytotoxic function; however, the relevancy of these findings to in vivo exposure remains unclear.

A.7 CARCINOGENESIS/ GENOTOXICITY

Summary: Carcinogenesis/Genotoxicity

During the initial review of atrazine in 1986, the Agency designated this pesticide as a Group C carcinogen ( i.e., possible human carcinogen) due to the increased incidence of mammary tumors seen in Sprague

- Dawley rats after chronic exposure and the lack of mechanistic data suggest ing a MOA that could be evaluated for human relevance. It should be noted that no other tumor sites were found in rats and there were no tumors induced in mice.

Since the 1986 assessment, an extensive mechanistic database has been generated by laborator ies across the world. The Agency presented its assessment of the available mode of action information to the FIFRA SAP in 2000 (FIFRA SAP, 2000). At that time it was concluded, that the data show that the operative MOA for mammary gland tumorigenesis invo lves the perturbation of the hormonal milieu through the HPG axis and not a genotoxic or mutagenic MOA. While the disruption of the LH surge that is central to atrazine’s MOA is a relevant endpoint to assess potential adverse reproductive effects, it is n ot relevant for mammary tumor development in humans. In rats, the mammary tumors arise as a result of constant stimulation of the mammary gland by elevated levels of circulating estrogen and prolactin during the early reproductive senescence caused by the disruption of the LH surge. In contrast, humans’ reproductive senescence (menopause) is not the consequence of disruptions at the hypothalamic/pituitary level, but rather a depletion of follicles and a decrease inPage 143 of 210 estrogen. That is to say, that while the hallmark of atrazine

- induced reproductive senescence in rats is elevated estrogen, in humans menopause is marked by a decrease in both estrogen and prolactin. Consequently, in the absence of constantly elevated estrogen and prolactin levels mammary gland tumors would not develop in humans as a result of atrazine exposure.

Several genotoxicity and carcinogenicity studies using transgenic and chemically

induced rodent carcinogenesis models have been published since the 2000 SAP review and the 2003 IRED. Researchers have studied atrazine’s potential effect on tumor progression in a number of glandular tissues (pituitary, thyroid, ovaries, and mammary gland). It should be noted that none of these experiments study the tumor

- initating capabilities of atrazi ne per se but rather its ability to accelerate the progression of already existing tumors. In general, these data show no evidence that atrazine exposure contributes to tumor progression or that it makes the tumors already present in the system more aggre ssive. In vitro studies

– for the most part

– corroborate the findings in the in vivo studies and show that atrazine is cytotoxic for most of the cell

lines tested.

Work by Fujimoto and coworkers in 2003 using transplanted MtT/E

- 2 pituitary rat cells (a cell line highly

- sensitive to estrogen

- induced proliferation), showed that in ovariectomized F344 rats exposed through the diet to atrazine at dose levels ranging from 5

- 500 mg/kg/day atrazine exposure did not accelerate tumor progression. In contrast, tu mors were observed within 9 days in animals treated with 0.5 mg subcutaneous pellet of estradiol. In this test system, serum prolactin levels, cell proliferation, and estrogen gene activation were also unaffected by exposure to up to 500 mg/kg/day of atra zine. Similarly, work by Son et al. (2003) using a DHPN

- induced thyroid carcinogenesis model demonstrated that atrazine exposure did not enhance thyroid tumor progression nor did it affect thyroid hormone levels (T 4 , T 3 , and TSH). DMBA

- induced ovarian carcinogenesis was yet another model used to study atrazine’s potential to affect tumor progression. Using this model, Tanaka et al. hypothesized that the operative mode of action leading to ovarian adenocarcinomas involv es hormonal disruption (particularly estrogen). Therefore, they postulate that exposure to atrazine (which has been associated with hormone disruption) may also affect ovarian carcinogenesis. As expected, a single exposure to 0.5% DMBA induced ovarian tu mor formation. Subsequent atrazine exposure through the diet at doses ranging between 5

- 500 ppm led to a slight, non

- statistically significant decrease in the incidence of ovarian tumors seen after DMBA exposure alone. When considered collectively, the d ata reported by Fujimoto, Son, Tanaka and co

- workers demonstrate that atrazine exposure did not accelerate tumor progression.

Two in vivo studies are available. Fukamachi et al. in 2004 reported that in transgenic rats carrying a human c

- Ha

- ras proto

- oncogene atrazine exposure enhanced DMBA

induced mammary tumor development. However, a dose

- response could not be established for this finding. Likewise, the incidence of mammary adenocarcinomas appeared to be increased but a dose

- response relationship w as not observed since the incidence at the low

- and mid

- dose was higher than in the high dose. Non

- transgenicPage 144 of 210 rats were also used in this study, however, the data for these animals was not included in the report. Furthermore, atrazine exposure in vitro d id not cause MCF

- 7 cell proliferation

– an estrogen receptor

- positive human mammary cancer cell line. Consequently, it is difficult to ascertain if the changes seen after atrazine exposure are truly related to an inherent carcinogenic property of the comp ound or if it is due to the genetic background resulting from the presence of a human proto

- oncogene in the cells.

Ueda et al. conducted a similar set of experiments in 2005 but instead of using a transgenic rat model they used a DMBA

- induced mammary ca rcinogenesis model. In this study the effect of atrazine exposure on the progression of DMBA

- induced mammary tumors in ovariectomized Sprague Dawley rats was investigated. When DMBA

- induced rats were examined, no clearly discernible correlation between at razine exposure and the number of tumor

- bearing rats at the end of the study ( i.e., atrazine exposure did not increase the incidence of rats bearing tumors) was observed nor did atrazine exposure affect the regression of palpable tumors observed after ova riectomy. Some statistically significant changes were reported by the authors but that is mainly due to the statistical analysis used. Exposure to 500 ppm atrazine by itself did not induce any mammary tumors.

Agency scientists i dentified a number of deficiencies in the conduct, interpretation, and reporting in both the Fukamachi and Ueda studies which are described in Appendix A. Nonetheless, these two sets of data suggest that atrazine does not induce mammary tumors in transgen ic rats or ovariectomized rats treated with a mammary tumor inducer thus highlighting the importance of the ovarian hormones in the development of tumors in the mammary gland.

In vitro data evaluating the cytotoxicity and cell proliferative properties i n a variety of cell lines indicate that atrazine is cytotoxic or causes decreases in cell proliferation. In general, these data

– like the in vivo data

– suggest that atrazine is not genotoxic or mutagenic.

Chinese Hamster ovary (CHO) and human fibrob last cell proliferation was decreased at relatively low levels of atrazine exposure (Kmetic et al. 2008 and Manske et al. 2004). However, data from Yi et al. (2010) suggests that atrazine and its metabolites are not cytotoxic to two steroidogenic cell li nes (H295R and JEG

- 3) as well as two non

steroidogenic cell lines (HT

- 22 and MCF

- 7).

Comet assay

– a sensitive and rapid method for DNA strand break detection in individual cells

– was used to study atrazine’s capability of inducing genotoxicity. Using this assay, Garaj

- Vrhovac et al. and Zeljezic (2004) concluded that Gesaprim (an atrazine formulation) induced genome damage. The limitation of these studies is that a formulation of atrazine was used and no information is provided to determine the percen tage of atrazine present in the test material. Thus, it is entirely possible that the DNA breaks reported after Gesaprim exposure may be related to other chemicals in the formulation rather than atrazine per se . Sister chromatid exchange (SCE) analysis

– a sensitive method used to study DNA instability or a deficiency in DNA repair

– has alsoPage 145 of 210 been used to study the genotoxic potential of atrazine in human peripheral lymphocytes arrested at the G2 phase of the cell cycle. Using this method, Malik et al. d emonstrated that over a concentration range of 5

- 200 μg/L atrazine did not increase the frequency of SCE induction in G2

- prematurely condensed chromosomes.

Finally, two studies intended to evaluate if vitamin E treatment following atrazine exposure would ameliorate atrazine

- induced effects are available. After atrazine exposure at a high dose of 300 mg/kg/day, Singh et al. (2008) report a marked increase in lipid peroxidation and decrease in acetylcholinesterase activity and cell membrane lipid content s uggesting an increase in oxidative stress. Concurrent exposure to atrazine and vitamin E dampened all of these effects. A subsequent study by the same research group showed that at 300 mg/kg/day of atrazine induced DNA instability as measured by the Come t and micronucleus assays. Again, vitamin E exposure at 100 mg/kg/day decreased micronuclei formation and DNA breaks. The results of this study are questionable given that the atrazine doses used to induce these genotoxic effects are orders of magnitude higher than what is currently used as the point of departure for human health risk assessment.

A.7.1 Fujimoto, N and Honda, H. (2003). Effects of Environmental Estrogenic Compounds on Growth of a Transplanted Estrogen Responsive Pituitary Tumor Cell Line in Rats. Food Chem. Toxicol. 41:1711

- 1717. The objective of this study was to evaluate the role genistein, p

- nonylphenol, or atrazine as tumorigenic promoters for estrogen

- sensitive tumors. A rat pituitary cell

- line

– MtT/E

2

– that is responsive to estrogen exposure was transplanted into ovariectomized rats. The rats were subsequently exposed to estradiol, genistein, p

- nonylphenol, or atrazine and examined twice a week for the development of tumors. 17 In addition, MtT/E

- 2 cell cultures were transfe cted with an estrogen

- response

- element

- luciferase reporter gene and evaluated for activation of the reporter gene. These cells were also grown in vitro and evaluated for cell proliferation after exposure to the chemicals under evaluation. Finally serum c ollected at the time of sacrifice was used to evaluate prolactin levels.

Six week old ovariectomized F344 rats (sample size not reported) were inoculated with MtT/E

- 2 tumors cells and subsequently exposed through the diet to 0, 5, 50, or 500 mg/kg atrazin e (purity not reported) for 33 days. Animals were examined for tumor development twice per week and sacrificed when the tumor size reached 1

- 2 cm in diameter. At necropsy, the pituitary gland and uterus were collected and weighed and blood serum obtained to measure for prolactin levels. For cell proliferation assessments, MtT/E

- 2 cell cultures were exposed to 0 or10 - 7

- 10 - 4 M atrazine for 7 days and cell proliferation measured using the WST

- 1 assay (reduction WST

- 1 into formazan dye by viable cells). Act ivation of the ERE

- luc reporter gene was evaluated by measuring luciferase activity in MtT/E

- 2 cells transfected with the ERE

- luc reporter gene and subsequently exposed to 0 or 10 - 7

- 10 - 4 M atrazine for 24 hours.

17 In this evaluation only the results for atrazine will be discussedPage 146 of 210 Atrazine exposure did not result in MtT/E

- 2 cell proliferation according to the WST

- 1 assay nor did it have an estrogenic response as evidenced by a lack of response in the ERE reporter gene assay. After 33 days of exposure, atrazine did not cause any changes in body, uterine, or pituitary weights . Serum prolactin levels were also unaffected by exposure to up to 500 mg/kg atrazine. In contrast, exposure to a 0.5 mg subcutaneous pellet of estradiol resulted in a marked decrease in body weight, and an increase in uterine and pituitary weights along with a marked increase in serum prolactin ( ↑7

- fold, p<0.01). Tumor development was also unaffected by treatment with atrazine with evidence of tumor development first appearing on day 24 of the experiment for animals in the control or atrazine treated gr oups as opposed to 9 days for animals treated with estradiol.

Reviewer’s Comments: The current study had a number of deficiencies including the lack of data on the purity of the atrazine used in the study or the sample size. Nonetheless, some general c onclusions may be drawn from this study. In particular, that atrazine exposure up to 500 mg/kg did not enhance the appearance of estrogen

dependent pituitary tumors nor does it result in increased cell proliferation. When considered collectively, the data reported by Fujimoto, Son, Tanaka and co

- workers demonstrate that atrazine exposure did not accelerate tumor progression.

A.7.2 Fukamachi, K., Han, B.S., Kim, C.K., Takasuka, N., Matsuoka, Y., Matsuda, E., Yamasaki, T., and Tsuda, H. (2004). Possible enhancing effects of atrazine and nonylphenol on 7,2

- dimethylbenz[a]anthracene

- induced mammary tumor development in human c

- Ha

- ras proto

- oncogene transgenic rats. Cancer Sci. 95(5):404

- 410.

This study evaluates the potential of atrazine or nonylphenol to promote growth of mammary tumors induced by 7,2

- dimethylbenz[a]anthracene (DMBA) in transgenic (Tg) rats carrying the human c

- HA

- ras proto

- oncogene. These rats are exquisitely sensitive to DMBA

- induced mammary tumors. 18

18 In this evaluation only the results for atrazine will be discussed Two

- hundred seventy six female and male Tg and non

- Tg rats were administered a single 25 mg/kg dose of DMBA via gavage at an age of 50 days. Beginning on the following day, animals were exposed in the diet to 0, 5, 50, or 500 ppm atrazine (purity unknown) [ ≈ 0, 0.55, 6.2, or 36.4 mg/kg/da y ( ♀) and 0, 0.26, 2.75, or 24.23 mg/kg/day ( ♂)]. The Tg females were exposed to atrazine for 8 weeks while the non

- Tg females and all males were exposed to atrazine for 20 weeks. At the end of the exposure period, animals were sacrificed by exsanguinati on, the numbers of grossly observed tumors were recorded and subsequently measured and subjected to histopathological examination. In addition, body, liver, kidney, ovarian, uterine, and testis weights were recorded. Levels of serum atrazine were measure d after necropsy by liquid chromatography and mass spectrometry (LC/MS). The potential impact of atrazine on cell proliferation was also measured by exposing the human mammary cancer cell line MCF

- 7 to atrazine for 3 days. Cell proliferation was then mea sured using the WST

- 1 assay (measures viable cells capability to reduce WST

- 1 into formazan dye).Page 147 of 210 Tg females exposed to 500 ppm atrazine exhibited a statistically significant decrease in body weight ( ↓14%, p<0.01) with a concomitant decrease in food consu mption ( ↓10%, p< 0.05). A statistically significant increase in the incidence of mammary adenomas ( ↑67%, p<0.01 vs 17% control) was noted following exposure of Tg female rats to 5 ppm atrazine. However, no increase was observed after exposure to 50 or 50 0 ppm atrazine. The incidence of mammary adenocarcinomas at 5 and 50 ppm atrazine was 100% (control: 50%) although the incidence at 500 ppm was lower ( ≈86%). In contrast to the apparent increase in mammary tumors in Tg females, Tg males exposed to 500 pp m atrazine had a decrease in the incidence of skin tumors (40% vs. 67% control) although a non

- statistically significant increase in the incidence of mammary adenocarcinomas (100% vs 56% control) was also reported at this dose level. No data were provided on non

- Tg rats (males or females).

The study authors concluded that atrazine appeared to enhance mammary tumors in female Tg rats but without any dose relationship while decreasing the incidence of skin tumors in Tg males. Furthermore, the authors con cluded that atrazine did not increase cell proliferation of MCF

- 7 cells. Several limitations were identified in this study that hinders a meaningful interpretation of the results. As an example, Tg female rats received atrazine for 8 weeks, the remainder of the animals in the study received atrazine for a considerably longer period (20 weeks). In addition, the purity of the atrazine used in the study was not reported, no exposure concentrations were provided for the MCF

- 7 experiment, and no data were pre sented for the non

- Tg rats or for the MCF

- 7 cell proliferation assay.

Reviewer’s Comments: The lack of data on non

- Tg rats is a critical deficiency in this study. In experiments conducted by Ueda et al. using non

- Tg rats treated with DMBA to induce mamm ary tumors, a relationship between atrazine exposure and increases in the incidence of tumor

- bearing rats cannot be drawn. Thus, in the current study the different treatment and lack of data on the non

- Tg rats precludes reviewers from drawing a conclusion on whether the increases seen in mammary adenocarcinomas were related to atrazine exposure. The lack of dose

- response on the incidence of mammary adenomas and adenocarcinomas further confounds the interpretation of these data.

A.7.3 Garaj

- Vrhovac, V. and Zeijezic, D. (2004) Genotoxicity evaluation of pesticide formulations containing alachlor and atrazine in multiple mouse tissues (blood, kidney, liver, bone marrow, spleen) by comet assay. Neoplasma 51.3:198

- 203.

In this study the authors evaluated po ssible genome damage caused by two pesticide formulations (Bravo® and Gesaprim®) containing alachlor and atrazine as active ingredients by applying the comet assay on mouse cells from blood, kidney, liver, bone marrow, and spleen. 1Page 148 of 210 The focus was intende d to uncover the possible genotoxicity of pesticide formulations and not only their active ingredients. Workers who handle these products during their production and application stages are exposed to the complex mixture contained in the formulation versus just the isolated active ingredients

– which in turn could give results that don’t correspond to the actual conditions.

Gesaprim®, containing 500 mg/ml of atrazine as the active ingredient were given at the doses of 1.08 ml/kg (540 mg/kg a.i.) and 0.07 μ l/kg (3.5 X 10 - 2 mg/kg a.i.) to five male CBA mice assigned to 4 treatment groups and a control group injected intraperitoneally once. The lower dose represents the oral chronic RfD and the higher dose administered is compatible with that dose which shows survival in mice in vivo . Methyl methanesulfonate (MMS) at 70 mg/kg was used as a positive control. The mice were sacrificed 24 hours after treatment; blood (taken into heparinized vacutainers) and selected organs were weighed, minced, and suspended in a chilled homogenization buffer.

The alkaline comet assay of blood samples was performed according to standard protocol, while organ samples were subjected to a modified assay (Sasaki, YF, 1997). Each organ and pesticide concentration, 100 cells were an alyzed by the Comet assay II automatic digital analysis system to determine tail length and tail moment. Any differences of assay endpoints between control blood and organs and those from treated mice were evaluated using the Mann

- Whitney U

- test.

Results showed no death, morbidity, or distinctive clinical signs observed after any treatment (including during necropsy). Mean values of the comet assay endpoints were found to be statistically significant (p<0.01) regarding an increase of tail length for all 5 tissues examined in mice in low (0.07 μl/kg) and high (1.08 ml/kg) treatment groups compared to control. The DNA of the kidney and liver, for both concentrations given, showed the largest migration. There was also a significant increase for all tissues in the higher concentration treated group when compared to the lower concentration group. For mice given the lower concentration, the tail length values of the kidney and liver were significantly higher than those of blood, bone marrow, and spleen from t he same group but non significant difference was found between the liver and kidney; while in the higher dose group the kidney and liver tail lengths were significantly higher but no statistical difference was found. Tail lengths in the kidney DNA from mi ce treated with 1.08 ml/kg Gesaprim® were up to 99 μm versus 84 μm for mice in the 0.07 μl/kg group (data not shown). For the liver, the tail lengths at the higher dose were 97 μm whereas at the lower dose it was 39 μm, much lower when compared to the kid ney.
The authors have concluded that regardless of the dose, genome damage in all tissues examined was detectable with the comet assay. The most affected organs were the kidney and liver. This paper also cites several studies which show contradictory re sults both in vitro and in vivo . This study also gave descriptions of studies on human lymphocytes showing no appearance of genotoxicity and provided a rationale as to why the toxic activity maybe slightly increased at higher doses when compared to the Rf D,Page 1 49 of 210 could be due to the removal of apoptotic cells (data not shown). Other apoptotic detection methods should be employed before presenting those results.

Reviewer’s Comments: Significant (p<0.01) increases in the tail length and tail moment were record ed for all tissues at both doses of atrazine. However, there are uncertainties regarding the atrazine findings. For example, the authors stated, “genome damage for both pesticides was found to be higher for the higher dose given”. It was noted, however, that the “dose response” is very flat considering that more than two orders of magnitude separate the atrazine doses that were tested. Nevertheless, the relevance of the DNA damage data for atrazine is uncertain because neither the species (rat) nor the target tissue for tumors (mamma ry tumors) were examined.

Overall, the use of the alkaline comet assay on different tissue samples could provide evidence of genotoxic effects of pesticides in vivo . Garaj

- Vrhovac et al. and Zeljezic and Garaj

- Vrhovac (200 4) concluded that Gesaprim (an atrazine formulation) induced genome damage. The limitation of these studies is that a formulation of atrazine was used and no information is provided to determine the percentage of atrazine present in the test material. Th us, it is entirely possible that the DNA breaks reported after Gesaprim exposure may be related to other chemicals in the formulation rather than atrazine per se

.

A.7.4 Kmetic, I., Srcek, G., Slivac, I., Simic, B., Knlewald, Z., Kniewald, J. 2008. Atra zine Exposure Decreases Cell Proliferation in Chinese Hamster Ovary (CHO

- K1) Cell Line. Bull Environ Contam Toxicol. 81: 205

- 209.

The purpose of this study was to determine the toxic effect of atrazine at the ovarian cellular level. The Chinese Hamster O vary (CHO

- K1) cell line was used as the test system for evaluation of in vitro atrazine cytotoxicity and the morphological changes were followed during cell death.

CHO

- K1 cells were treated with atrazine at concentrations ranging from 10

- 160 μg atrazine/m L of cell suspension (46

- 742 μM) for 24, 48 or 72 hours. The cell viability was determined by four different bioassays: Trypan blue exclusion method, Kenacid Blue, Neutral red and MTT assays. Trypan blue exclusion method was used to estimate the proportion of viable cells. The Kenacid blue assay determines the total cell protein content. The Neutral red uptake assay determines the accumulation of the neutral red dye in the lysosomes of viable uninjured cells. The MTT assay determines the viability of cel ls.

All four methods showed clear dose

- dependent cytotoxic effects. Concentrations of atrazine 20

- 80 ug/mL followed by Trypan blue method showed significantly decreased proliferation of cells in CHO

- K1 cells line at all dose levels, with the toxicity pro gressively increased with high atrazine concentrations. After 72 hours incubation period, the 20 ug/mL atrazine concentration significantly decreased cell proliferation byPage 150 of 210 19% in CHO

- K1. Cell viability was decreased 49% at 60 ug atrazine/mL and 55% at 80 ug atrazine/mL, both at p<0.001.

IC 20 and IC 50 values were determined after 24, 48 and 72 hours for the four methods. A comparison of the IC 20 values after 48 hours or IC 20 and IC 50 after 72 hours of incubation showed that Neutral red, Kenacid blue and M TT assays showed a similar rate of toxicity. The Trypan blue assay was more sensitive. The IC 50 value for trypan blue after 72 hours was two times lower than the IC 50 s for the other 3 assays. Morphologically, the level of apoptosis in atrazine treated ce lls was low.

Reviewer’s Comments: Cell proliferation was decreased in Chinese Hamster ovary (CHO) cell lines at relatively low levels of atrazine exposure.Page 151 of 210 A.7.5 Lasserre, JP, Fack, F, Revets, D, Planchon S, Renaut J, Hoffmann L, Gutleb, Muller CP, Bo hn T, (2009) Effects of the Endocrine Disruptors Atrazine and PCB 153 on the Protein Expression of MCF

- 7 Human Cells. Journal of Proteome Research.

The present study aims to investigate the proteomic effects of endocrine disrupting compounds using MCF

- 7 cells as a model. MCF

- 7 cells were chosen as these cells are derived from a human breast cancer and their growth is being stimulated by 17

- β estradiol. To identify changes of protein expression in MCF

- 7 human cells, they were treated with either 100 ppb at razine or PCB153 during 24 h. A negative control containing DMSO was included as well as a positive control containing 17

- β estradiol. Proteins from the two groups (Atrazine, PCB153) and two controls (positive and negative controls) were separated by isoelectric focusing (IEF) followed by SDS

- PAGE. After separation and analysis, the differentially expressed protein spots were excised, digested by trypsin, analyzed by MALDIToF and identified by comparison with databank searches.

The chemicals and mate rials used consisted of Atrazine (98% purity), PCB153 (100% DMSO with 2mg/mL stock solution), 17

- β estradiol (DMSO 10uM solution), cell cultures were done in monolayers (MCF

- 7bus cells), protein extraction and analysis were performed and used to obtain su fficient biological material. Protein labeling was done with a pooled “internal standard” and were labeled prior to mixing and electrophoresis with CyDyes. Bidimensional electrophoresis was also performed. Labeled protein images were then captured and a nalyzed by excitation of gels. Proteins were then identified by searching against the NCBInr databases using GPS Explorer Software v3.6. The results were displayed in showing a total of 5,828,094 sequences of which 272,706 were for humans. The search pa rameters allowed fixed modifications for cysteine and methionine and variable modifications of tryptophan (kynurenin, oxidation and double oxidation) as well as peptide N

- terminal E or Q as pyroGlu. All identifications were manually verified and validated . Only the proteins having a protein score superior to MASCOT protein score threshold for significant homology were considered. The acceptance criteria allowed for ion scores greater than 26 or 43 indicate homology and identity, respectively.

The results for m orphology and adhesion of MCF

- 7 cells showed differ ent observations between the four conditions indicated that the different treatments had an effect on morphology and adhesion of cells. Cells exposed to 17

- β estradiol were spread out, whereas the c ells were more rounded and comparatively smaller for the atrazine, PCB153 and DMSO treatment. The analysis of the different treatments revealed that overall and within all pairwise comparisons, 66 spots were differentially regulated in their expression ( P < 0.05, 2

- sided).

From these 66 protein spots, 49 were tentatively identified, belonging to 29 different proteins. Out of these, 22 different proteins were identified by at least two peptides, with a sufficiently high ion score. Some proteins (far upstre am element binding protein, histone H1b, actin, heat shock 70 kDa protein 8) were identified in more than one spot,Page 152 of 210 suggesting the presence of isoforms and/or post

- translational modifications that affect the p I and/or the molecular mass of the protein.

I dentified proteins were classified with respect to their subcellular localization and biological processes, according to information collected on Swiss

- Prot (http://www.expasy.ch/sprot/). The majority of the identified proteins were intracellular proteins (88.6%), mostly distributed in the nucleus (34.3%) and cytosol (34.3%). Concerning the function of the proteins of interest, results showed that the proteins identified were involved in various processes. The e ffects of atrazine and PCBs on the Proteome o f MCF

- 7 Human Cells showed that a mong the 22 differentially expressed proteins, 10 were specifically differentially expressed upon atrazine treatment, 4 were specifically differentially expressed following the PCB treatment and 8 were both differentially e xpressed after atrazine and PCB treatment. Among 18 differentially expressed proteins during the atrazine treatment, 88% were underexpressed, and among 12 differentially expressed proteins during the PCB treatment, 75% were overexpressed. These results in dicate that atrazine treatment seems to decrease the activity of the cells, whereas the PCB153 treatment stimulated the cells.

The authors concluded that this is the first report on the effects of atrazine and PCB153 at the proteomic level on MCF

- 7 human cells, a cancer cell line. They revealed a total of 22 differentially expressed identified proteins, predominantly involved in transcription processes, stress regulation and structural components. They claim that their findings indicate that atrazine tre atment seemed to decrease the activity of cells, whereas the PCB153 treatment stimulated the cells. The present findings indicate that the differentially expressed proteins were not located in a single compartment of the cell. Furthermore, different func tions of the cell were affected, indicating that atrazine targeted neither a unique compartment nor a single process in the cell. The causes are purely speculatory.

The observed expression levels of these proteins following atrazine and PCB153 exposure may have contributed not only to DNA damage as previously described for PCB153 or atrazine but also to changes of the expression level of certain genes. The present study aimed to understand the mechanism of action of atrazine and PCB153 on MCF

- 7 human c ells in particular. Here, it was ultimately found that atrazine and PCB153 have effects on the expression of several proteins located in different cell compartments and are involved in various processes such as oxidative stress, DNA damages, shape of the cell, gene expression regulation or spermatogenesis. It is suggested that future studies should target cellular subfractions such as membrane, cytosol and nucleus. Also performing studies which are designed to go into further detail with various concentra tions of additional individual EDC compounds would be useful. Since the environment is most often polluted by several pollutants that could act in synergy it would be prudent to study the effects of mixtures.

A.7.6 Malik, S. I., Terzoudi, G.I., and Pant elias, G.E. (2004). SCE analysis in G2 lymphocytes prematurely condensed chromosomes after exposure to atrazine: the non

- dose

- dependent increase inPage 153 of 210 homologous recombinational events does not support its genotoxic mode of action. Cytogenet Genome Res 104 : 315

- 319.

Human peripheral lymphocytes, collected from seven healthy volunteers, were grown, stimulated with phytohemagglutinin and exposed to atrazine (unreported purity) concentrations ranging from 5 to 220 μg/mL atrazine, the solvent (dimethyl sulfoxi de ) or the positive control (mitomycin C) for 24 hours. Three separate experiments were conducted. Prior to the end of treatment, calyculin

- A was added to the appropriate cultures for the last 1 hour of incubation (to arrest cells at G2) or colcemid for the last 3 hours [to arrest cells at metaphase (M

- phase)]. The rationale for using cell arrest at G2 rather than the conventional approach of colcemid treatment to collect cells at metaphase was to determine whether atrazine would increase the frequency of homologous recombination, a DNA repair process associated with SCE induction. Cells were collected, stained by the fluorescent

- plus

- Giemsa technique and 30

- 50 cells were scored for each culture; triplicate cultures were prepared per treatment group.

R esults from the first experiment showed that concentrations ≥220 μg/mL atrazine could be scored for SCE induction in G2

- phase cells while no metaphases were found at 120 and 220 μg/mL in the colcemid

- treated cultures. Thus, SCEs could be scored even at le vels that were lethal in the conventional assay and there was no significant increase in SCE induction at 60

- 220 μg/mL. Results for the second experiment were in good agreement with the first trial and indicated that atrazine over a concentration range o f 5 to 200 μg/mL did not increase the frequency of SCE induction in G2

- prematurely condensed chromosomes (PCCs). Using cells treated with 20 μg/mL and arrested with either colcemid or calyculin

- A, trial three results indicated that the variation in SCE f requencies in lymphocyte cultures is significantly lower in G2

- PCCs than in SCEs scored in the metaphase cells.

Reviewer’s Comment: The assay was well

- conducted and indicates that there was convincing evidence that atrazine is not genotoxic in this test system.

A.7.7 Manske, M., Beltz, L, and Dhanwada, K. 2004. Low

- Level Atrazine Exposure Decreases Cell Proliferation in Human Fibroblasts. Arch. Environ. Contam. Toxicol. 46:438

- 444.

This purpose of this study was to examine the effect of low

- level, shor t

- term atrazine exposures on human fibroblast cells in culture by assessing cell proliferation and apoptosis.

Normal human fibroblasts, DET 551, were used in the saturation density analysis to measure cell proliferation. In this assay, DET 551 cells were plated at 2x10 5 cells/100 mm dish in MEM with 0.5% FBS (low serum medium). Cells were incubated at 37oC with 5% CO 2 for 48 hours in the low serum medium to synchronize cells. After 48 hour the medium was removed and replaced with complete medium with 10% FBS containing increasing concentrations of atrazine or DMSO (0, 3, 10, 50, 100, 200, and 300 μg/L). Atrazine dilutions were made from an atrazine stock concentration of 32Page 154 of 210 mg/L dissolved in DMSO. Cells were grown for 5 days at 37oC and 5% CO 2. After harv esting, individual cells were counted by the trypan blue exclusion method. Viability of cells in all treatment groups and controls was over 99%. The actual number of cells treated with increasing concentrations of atrazine or solvent control was compared to the number of control cells. Cells were counted using a hemocytometer. All experiments were run in triplicate.

An MTT assay was run to confirm the results for cell proliferation. This method does not rely on individual cells being counted. It is a calorimetric assay. DET 551 cells were plated in flat

- bottom 96

- well tissue culture treated plates at 2000 cells/well i n low serum medium and incubated for 48 hours. After 48 hours, the medium was removed and replaced with complete medium with 10% FBS and increasing concentrations of atrazine or DMSO (0, 3, 10, 50, 100 and 200 μg/L) for an additional 48 or 72 hours . The

a trazine stock concentration was the same as before, 32 mg/L in DMSO. For MTT analysis, 20 μl of 5 mg/ml MTT in PBS was added to cells and plates were incubated for 4 hours at 37oC. HCl in isopropanol was added to each well, and optical density was read on a UV/VIS spectrophotometer. A similar procedure as followed for human peripheral blood mononuclear cells (PBMCs), composed of lymphocytes and monocytes. One million PBMCs were plated in 24 well tissue culture plates in PRMI with 5% FMS containing increasi ng concentrations of atrazine or DMSO (40, 100 or 200 μg/ml). Cells were incubated for 96 hours at 37 oC and 5% CO 2. Then 150 μl of MTT solution was added and cells were incubated for an additional 4 hours at 37oC. HCl in isopropanol was added to each wel l, and optical density was read at 570 nm. The increase in OD had a positive correlation with viable cell number and indicated enhanced cell growth compared to control cells. All experiments were run in triplicate.

To determine if the decrease in cell nu mber was due to apoptosis of the treated cells, two assays were done

— a qualitative immunohistochemical analysis and a quantitative enzymatic analysis (caspase assay). In the qualitative immunohistochemical analysis, apoptotic cells are detected with a biot in

- streptavidin system complexed with horseradish peroxidase and a diaminobenzidine

- based stain. DET 551 cells were exposed to atrazine or DMSO at concentrations of 0, 3, 50 or 200 μg/L for 48 hours before the assay was performed. Cells treated with TACS n uclease was the positive control. The Caspase assay was used to measure apoptosis enzymatically. Caspase

- 3 activity in cell extracts was measured using the CaspASE colorimetric assay system protocol. The CaspASE assay is a colorimetric assay that measure activity of caspase

3. Cytosolic thiol proteases called caspases are key enzymes involved in apoptosis. DET 551 cells were exposed to increasing concentrations of atrazine or DMSO (0, 3, 100, 200, and 500 μg/L) for 48 hours before analysis.

Cell Prolife ration Results : The saturation density analysis showed that DET 551 fibroblasts exposed to increasing concentrations of atrazine showed a decrease in cell growth compared to control cells. Cells exposed to 50, 200 and 300 μg/L atrazine had a statistically significant decrease (1.2

- 1.5 fold) in cell number compared to control cells (p<0.05). The authors also noted a decrease (not statistically significant) in cell growth in the highest concentration of DMSO (300 μg/L) compared to the other DMSO controlPage 155 of 210 val ues. This could indicate that this concentration of DMSO may be slightly toxic by itself since the percentage of DMSO in these cells is higher than in the other samples and these cells were exposed to the chemical for 5 days, 2 days longer than in the MTT analysis.

In the MTT analysis, a 48 hour exposure to atrazine showed a statistically significant decrease (p<0.05) in DET 551 cell proliferation. Cells exposed to 10

- 200 μg/L had a dose related significantly decreased absorbance (1.1

- 1.7 fold) compared to controls, indicating a decrease in cell proliferation. There was no statistically significant decrease in proliferation in solvent

- control

- treated cells. There was no effect on cell proliferation by MTT analysis when human PBMCs were treated with incr easing concentrations of atrazine for 96 hours.

Apoptosis Results : In the immunohistochemical analysis, DET 551 cells were exposed to 0, 3, 50, or 200 μg/L atrazine or DMSO for 48 hours before the assay was performed. The atrazine and DMSO

- treated sampl es were compared qualitatively to a TACs nuclease

- positive control. When the cells treated with 200 μg/L atrazine or DMSO were compared to control cells, there was only a sight increase in nuclear staining of atrazine

- treated cells, however, the level of a poptosis in treated

– treated cells was low compared to the positive control. The caspase assay was then done to determine if the small increase in the apoptotic cells was significant. The results showed that all atrazine

- treated samples (3

- 500 μg/L) had cas pase activity (7.31

- 9.53 pmol/μl) similar to the negative control (7.31 pmol/μl). No significant differences were detected between the control treatments (DMSO) and atrazine

- treated cells.

Conclusion: In vitro exposure to low levels of atrazine (as low as10 μg/L) for a short exposure (48 hours) causes a decrease in cell proliferation in human fibroblasts but not in PBMCs. This study showed that the decrease in cell proliferation of human fibroblasts was not attributed to an increase in apoptosis.

Review er’s Comments: In vitro data evaluating the cell proliferative properties in a variety of cell lines suggest indicate that atrazine causes decreases in cell proliferation. In general, these data suggest that atrazine is not genotoxic or mutagenic. Mansk e et al. 2004 showed that human fibroblast cell proliferation was decreased at relatively low levels of atrazine exposure.

A.7.8 Mastrangelo, S., Tomassetti, M, Carratu, M.R., Evandri, M.G., and Bolle, P. (2006). Quercetin reduces chromosome aberrations induced by atrazine in the Allium cepa test. Environ and Mol Mutagen 47:254

- 259.

In this study, groups of six peeled, white commercial bulbs of Allium cepa, also known as the “garden onion”, were placed in glass tubes containing mineral water and the fl avonoid, quercetin (Q), which is thought to have antioxidant properties, was tested at 0.1 to 20 μg/mL Q. Bulbs were also exposed to atrazine (unreported purity) at 0, 2.5, 5.0 or 7.5 μg/ml and varying combinations of Q and atrazine (0.5 or 5 μg/mL Q with 2.5,Page 156 of 210 5.0 or 7.5 μg/mL atrazine), and incubated in the dark for 5 days. Test solutions were renewed daily and “topped

- up” on days 3, 4, and 5. Ten replicate bulbs per each treatment group were prepared. Bulbs were observed daily throughout the 5

- day exp osure period for root length and form and the presence of “crochet hooks” and tumors. At the end of treatment, the meristematic cell (analogous to stem cells in mammalian tissue) region from the root tips was harvested and processed for cytogenetic evalua tion. One thousand cells were counted to determine the mitotic index (MI) and a total of 400 metaphases and 400 ana

- /telophases (40 metaphases /slide and 40 ana

- /telophases/slide) were scored for each treatment group.

No effects were observed on root m orphology after exposure to Q or atrazine. Similarly, Q alone did not affect the MI at any concentration. By contrast, atrazine alone caused an 18% reduction in the MIs at the highest level tested (7.5 μg/mL); no cytotoxic effects were noted at lower dos es. Combined with either 0.5 or 5 μg/mL Q, 7.5 μg/mL atrazine affected a slightly lower reduction in the MI (~23% ↓ for both Q concentrations) then when the pesticide was tested alone. Neither Q nor 2.5 or 5.0 μg/mL atrazine were clastogenic; however, 7. 5 μg/mL atrazine induced a significant (p<0.001) increase in structural chromosome aberration. The most prominent type of aberration was fragments. When the plant cells were assayed with 0.5 μg/mL Q in combination with 7.5 μg/mL atrazine, the frequency o f chromosome aberrations was listed as significantly (p<0.05) lower (~43% ↓) compared to 7.5 μg/mL atrazine alone. However, no increased protection was afforded by the higher concentration of Q.

Reviewer’s Comments: The study was well

- conducted and the se data provide evidence of a clastogenic response induced by atrazine at 7.5 μg/mL atrazine, a concentration resulting in adequate cytotoxicity (~ 20% ↓in the MI). The data also suggest that the clastogenic activity is likely to be confined to a narrow r ange of doses because concentrations >7.5 μg/mL atrazine would likely exceed the recommended level of cytotoxic and no cytotoxicity or clastogenicity was noted at ≤ 5.0 μg/mL.

A.7.9 Olejnik, Am, Marecik, R, Bialas, W, Cyplik, P. (2010). In vitro studies on atrazine effects on human intestinal cells. Water Air Soil Pollut. DOI 10.1007/s11270

- 010

- 0394

- 0; published inline: April 16, 2010

In an in vitro study, cultured Caco

- 2 cells from the established Caco

- 2 cells line, which is a continuous line of het erogeneous human epithelial colorectal adenocarcinoma cells, were exposed to atrazine (purity not specified) concentrations of 0, 1, 10, 50, and 250 μM, prepared in dimethyl sulfoxide (DMSO). A series of experiments were carried out to simulate long

- term exposure (cells treated with atrazine levels ranging from 1

- 250 μM for 7 days or continuously treated with 10 μM for 4 weeks) and short

- term exposures (cells cultured for 7, 14 or 21 days were harvested and exposed to 50 and 250 μM atrazine for 3 hours). Six replicate cultures were prepared for the negative control (culture media alone), the solvent control (0.1% DMSO), and the treatment groups. After exposure, treated cells were monitored for viability, proliferation, differentiation, monolayer integri ty and DNA damage.Page 157 of 210 Results were as follows for the key endpoints that were measured ( i.e., cytotoxicity and genotoxicity):

Cytotoxicity

For the long

- term exposure, cell viability and cell growth (indicated by generation time) were unaffected by treatment with concentrations as high as 250 μM for 4 days. After 5 days of treatment with concentrations ≥50 μM, however, cell survival was reduc ed 17 and 40 % and cell generation time was increased from a background level of 46 hours to 52 or 64 hours for cultures dosed with 50 or 250 μM, respectively. In support of the above observations, decreased cell viability and cell density were clearly ti me

- related as indicated by the marked and significant reduction in the cell viability and density of cultures treated chronically with 10 μM atrazine (decreases of ~20, 40 and 80% after 14, 21 or 28 days, respectively). Mitochondrial activity was also ass essed as an indicator of viability, and the investigators found that metabolic activity was significantly altered in cultures treated with 250 μM for 14 and 21 days (~30 and 20 % reduced, respectively, compared to control). In addition, alkaline phosphata se (AP) activity was measured to determine enterocytic cell differentiation (an indicator of the activity of microvilli within the entrocytic surface). These data show that at 10, 50 or 250 μM, AP activity was 2.5, 4.4, or 10.2

- fold lower than control, respectively. Based on the evidence of significant and dose

- related effects on AP activity, the investigators concluded that the inhibition of intestinal cells maturation could be the main consequence of the long

- term exposure to atrazine. The final test conducted to assess cytotoxicity was the transepithelial electrical resistance (TEER) test; this procedure is designed to determine the effects of a test substance on the integrity of the monolayer as an indicator of the integrity of the intestinal epithel ial barrier. In agreement with the data from the other assays, the investigators found that 50 and 250 μM atrazine had a “strong effect” on the “intestinal cell monolayer development and confluence”.

For the acute exposure, the low atrazine concentration (50 μM) had no adverse effects on cell viability in cultures grown for up to 21 days prior to the 3

- hour exposure. At 250 μM, 7

- day old cultures were also largely unaffected. However, as the cultures differentiated and aged, viability dimin ished by ~5

- 10% in 14 and 21

- day old cultures.

Genotoxicity

Caco

- 2 cell cultures, grown for 7, 14 or 21 days were treated with 0, 50 and 250 μM atrazine for 24 hours, harvested and subjected to single cell gel electrophoresis (Comet test). Signific ant DNA damage was observed at all time intervals but only at the highest concentration tested.

Reviewer Comment: In general the study was well

- performed and the data support the study authors’ conclusions. Overall the data indicate that in this in vitr o study, acute exposure to atrazine concentrations up to 250 μM was not cytotoxic and only minimal cytotoxicity (~5

- 10% ↓ in survival) was seen in older cultures. By contrast, cytotoxicity,Page 158 of 210 manifested as reduced cell viability and density, delayed growth generation, reduced metabolic activity, and reduced monolayer integrity occurred in 14

- and 21

- day cultures treated with 50 or 250 μM (equivalent to 10 or 54 μg/mL). Significant damage to DNA in cells grown for 7, 14 or 21 days was only observed at the hi ghest concentration; no DNA damage was induced at 50 μM. The investigators claimed, however, that the cytotoxicity and DNA

- damaging activity of atrazine occurred at concentrations that would not be encountered in the environment. Hence, the relevance of these findings to the risk assessment is not clear.

A.7.10 Singh, M, Sandhir, R, Kiran, Ravi. (2010). Effects on antioxidant status of liver following atrazine exposure and its attenuation by vitamin E. Exp and Toxicol Pathol doi:10.1016/jetp.2010.01.005

Singh et al. (2010) investigated the potential of the non

- enzymatic antioxidant, vitamin E to inhibit oxidative stress induced by atrazine. Accordingly, male Wistar rats (6/group/sacrifice time) received single oral administrations of the vehicle (1 mL corn oil), atrazine (97.83%) alone (300 mg/kg in corn oil), vitamin E (100 mg/kg in corn oil) or atrazine + vitamin E ( 300 mg/kg atrazine + 100 mg/kg vitamin E). Animals were sacrificed 7, 14 and 21 days post treatment; livers were harvested, homogenized and samples were used to measure lipid peroxidation (LP), reduced glutathione (GSH), superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH

- Px), glutathione

s

- transferase (GST) and glucose

- 6

- transferase (GTS). Body and liver weights were als o recorded at each sacrifice interval.

Body weight of male rats receiving atrazine alone was significantly lower that control at all sample times; percent weight decreases ranged from

- 15% at 7 days to

- 26% at 21 days. By contrast, animals dosed with a trazine and vitamin E (A+VE) showed a weight loss at 7 days that was ~50% less then the atrazine only group. By 14 days, a significant (17%) increase in body weight was recorded for the A+VE group, which continued until the terminal 21

- day sacrifice. Liv er weights were consistently lower than control for the atrazine group and showed a 17% reduction at 14 days and a significant reduction at 21 days. Liver weights of the A+VE group were largely unaffected by treatment with atrazine. These finding suggest that vitamin E diminishes the toxic effects of atrazine on body and organ weight.

Similar evidence of the protective capacity of vitamin E toward atrazine

- induced liver damage was found in the data showing that the significant increases in LP and signi ficant decreases in GSH and G

- 6

- P D, which were generally evident by day 7, were lessened when vitamin E was included in the dosing regime. The adverse effects of atrazine on the antioxidant defense system of the intact animal, as indicated by the increas ed SOD, catalase, GPx, and G6PD activity in the livers of atrazine

- treated rats, which were clearly diminished in the presence of vitamin E, supports this assessment.

Reviewer Comment : In general the study was well

- performed and the data support the st udy authors’ conclusions that atrazine causes oxidative damage and alters thePage 159 of 210 “antioxidant status of the liver”. They further conclude that the administration of vitamin E along with atrazine ameliorates the toxic effects of atrazine.

A.7.11 Singh, M., Sandhir, R., and Kiran, R. 2008. Atrazine

- Induced Alterations in Rat Erythrocyte Membranes: Ameliorating Effect of Vitamin E. J. Biochem. Molecular Toxicology. Vol 22, No. 5: pgs. 363

- 369.

This study evaluated the possible beneficial effects of vitamin E against atrazine

induced oxidative stress in erythrocytes. Erythrocytes are susceptible to oxidative damage and a good model to understand oxidative damage induced by xenobiotics. Vitamin E is an antioxidant.

Male Wistar rats (n=18 animals/group) were ad ministered atrazine (300 mg/kg/day, 97.83% purity), Vitamin E (100 mg/kg/day), and combined atrazine (300 mg/kg/day)/Vitamin E (100 mg/kg/day) for a period of 7, 14, and 21 days. A vehicle control group was similarly treated. Blood samples were collected a t the end of the treatment periods. Erythrocyte membranes were prepared and analyzed for acetylcholinesterase (AChE) activity, lipid peroxidation (LPO), and lipid composition (total lipid content, phospholipid phosphorus, cholesterol). Protein content was also determined. Scanning electron microscopy (SEM) was used to examine morphological alterations of erythrocytes.

Atrazine treatment resulted in significant (p<0.05) decreases in AChE activity in erythrocytes at 7 days (22.32%), 14 days (25.58%), and 21 days (26.96%), compared to controls. Atrazine/Vitamin E treatment resulted in significant increases (12

- 14%) in AChE activity at 7, 14, and 21 days compared to atrazine alone treated animals.

Atrazine treatment resulted in a significant increase (p<0.05 ) in lipid peroxidation (nmol MDA/mg protein) compared to controls, with the effects being more pronounced with increasing duration. After 21 days treatment there was a 218.29% increase in MDA levels. The increase in LPO suggests that oxidative stress is i nduced by atrazine. Vitamin E treatment alone significantly (p<0.05) decreased MDA levels compared to controls. Atrazine/Vitamin E treatment significantly (p<0.05) decreased lipid peroxidation when compared to animals treated with atrazine alone.

Atrazine treatment resulted in a significant (p<0.05) decrease (24%) in the total lipid content of erythrocyte membranes only after 21 days, compared to controls. Vitamin E along with atrazine treatment significantly increased total lipid content to normal levels after 21 days, as compared to the atrazine treated group. Atrazine treatment significantly (p<0.05) decreased cholesterol content after 14 days (20.12%) and 21 days (32.94%). Atrazine/Vitamin E treatment significantly increased cholesterol content after 14 days (33.86%) and 21 days (57.02%), compared to the atrazine treated group. Atrazine treatment resulted in a significant decrease in phospholipid content after 14 days (16.76%) and 21 days (37.20%), compared to controls. Atrazine/Vitamin EPage 160 of 210 treatment s ignificantly increased phospholipid content after 14 days (22.86%) and 21 days (57.92%), compared to atrazine treated group.

Erythrocytes of rats treated with atrazine showed significant morphological changes as compared to the control group. These chang es included mild to moderate distortion in shape, significant ruptured membranes, and echinocyte formation, central or peripheral protuberances. SEM of erythrocytes of atrazine/vitamin E group shows improved erythrocyte topography as compared to atrazine

- t reated group. Extent of protuberance significantly decreased as compared to the control group.

Reviewer’s Comments: The results of this study are questionable given that the atrazine doses used to induce these genotoxic effects are orders of magnitude hi gher than what is currently used as the point of departure for human health risk assessment.

A.7.12 Singh, M., Kaur, P., Sandhir, R., Kiran, R. (2008). Protective effect of vitamin E against atrazine

- induced genotoxicity in rats. Mutat Res 654:145

- 14 9.

The protective effect of an antioxidant, vitamin E, against atrazine

- induced genotoxicity was investigated in young (~4

- 5 weeks old, based on the weight range of 100

- 120 g) 19 a Wistar male rats dosed with vitamin E alone, atrazine (97.83%) alone or a combination of atrazine and vitamin E in the comet and micronucleus assays. Accordingly, groups of six male rats were administered oral gavage doses of corn oil, 100 mg/kg/day vita min E, 300 mg/kg/day atrazine or a combination of comparable doses of vitamin E and atrazine for 7, 14 or 21 days. At the end of treatment, peripheral blood and liver were collected simultaneously from each test animal, processed and assayed for micronucl eus induction and/or DNA damage (gel electrophoresis and Comet assay).

Results from the gel electrophoresis on DNA isolated from the liver show that atrazine alone induced DNA damage as indicated by “shearing of the DNA” at 7, 14 and 21 days; the maximum effect was observed at 14 days. The investigators stated that atrazine did not induce the classic DNA ladder pattern, which is associated with apoptosis but shearing of DNA suggested DNA damage to the atrazine

- treated livers. Similarly, significant (p<0 .001) increases in the DNA tail length and tail moment were obtained from the comet assay performed on both blood and liver sampled at all harvest times. The response was generally time

- related and in good agreement with the electrophoresis data showing a marked increase in DNA damage at 14 and 21 days. By contrast, vitamin E alone was not genotoxic in either tissue. However, significantly (p<0.001) reduced (23

- 56% ↓) DNA tail lengths liver and blood samples were obtained for the tissues treated with atra zine plus vitamin E, compared to atrazine treatment alone. In agreement with these data, the results of the liver cell micronucleus assay demonstrated that atrazine alone increased (p<0.001) the frequency of micronuclei at all sample times and the respon se was time

- related with the largest increase occurring at 21 days. The treatment of the rats with atrazine (300 mg/kg/day) plus vitamin E (100

19 According to personal communications with Charles Parady, Senior Specialist, Charles River, male Wistar rats in the weight range of 100

- 120 gm would be approximately 4

- 5 weeks old.Page 161 of 210 mg/kg) caused significant (p<0.001) reductions in the frequency of micronuclei at 7 (23% ↓) and 21 (14%↓) days a s compared to atrazine

- treated rats.

Reviewer’s Comments: The results of this study are questionable given that the atrazine doses used to induce these genotoxic effects are orders of magnitude higher than what is currently used as the point of departure for human health risk assessment.

A.7.13 Son, H

- Y., Nishikawa, A., Okazaki, K., Lee, K

- Y., Imazawa, T., and Hirose, M. (2003). Lack of Modifying Effects of Atrazine and/or Tamoxifen on Thyroid Carcinogenesis in Rats Pretreated with N

bis(2

- hydroxypro pyl)nitrosamine (DHNP). Food Chem. Toxicol. 41:1811

- 1816.

In this study the researchers investigated the effect of atrazine and/or tamoxifen on the progression of DHPN

- induced thyroid carcinogenesis. 20 The goal of the study was to assess the impact of at razine (with or without concomitant tamoxifen exposure) on thyroid hormone changes and thyroid lesions induced by DHPN.

Twenty male F344 rats per group received a subcutaneous injection of 2800 mg/kg DHPN or saline. Starting one week later, the animals w ere exposed to 0, 5, 50, or 500 ppm atrazine (purity unknown) through the diet for 24 weeks. Rats receiving the saline injections were exposed to 0 or 500 ppm atrazine. At the end of the exposure, animals were sacrificed and the thyroid, pituitary, liver , lungs, kidney, pancreas, testis, epididymis, and brain were collected, weighed and subjected to histopathological examination. In addition, serum from 5 rats/group (except the 5 and 50 ppm atrazine groups) was collected and used to measure thyroid hormo nes (T 3 , T 4 , and TSH).

Exposure to 500 ppm atrazine resulted in a decrease in body weight ( ↓12

- 14%, p<0.05) regardless of DHPN exposure. The relative brain, liver, testicular, and epididymal weights from rats receiving both DHPN and 500 ppm atrazine were increased by ≈9

- 13% (p<0.05) when compared to animals receiving DHPN only. Moreover, when these rats were compared to the control group (no DHPN or atrazine exposure), the changes in body weight and relative organ weights were similar. Comparison of rats exposed to 500 ppm only (no DHPN injection) revealed that these animals experienced a 14% decrease in body weight (p<0.05) along with an 11

- 12% increase in relative brain, testicular, an epididymal weight. However, unlike pre

- exposure to DHPN follow ed by 500 ppm atrazine, exposure to 500 ppm atrazine alone did not elicit an increase in the relative liver weight. Histopathology assessments did not reveal any compound

- related changes though the data were not provided in the study report. Hormone anal ysis revealed that while there is a statistically significant decrease in T 4 levels ( ↓16%, p<0.05) after exposure to DHPN + 500 ppm atrazine relative to exposure to DHPN alone and no changes in T 3 or TSH. When compared to the control group, the decrease in T 4 is ≈10% and non

- statistically significant. Furthermore, exposure to 500 ppm atrazine alone did not elicit changes in any of the thyroid hormones while exposure to DHPN by itself resulted in a 7% increase in T 4 . This suggests that the apparent

20 In this evaluation only the results for atrazine will be discussedPage 162 of 210 decre ase in the hormone after exposure to DHPN + atrazine may be related to the increase above background caused by DHPN and not indicative of an effect of atrazine on thyroid hormone homeostasis.

Reviewer’s Comments: The results of this study indicate that at razine does not have an impact on thyroid carcinogenesis or thyroid hormone homeostasis. With the exception of a decrease in body weight, atrazine did not appear to have any effects on the parameters evaluated in this study. No histopathological changes were reported after exposure to 500 ppm atrazine and the increases in relative organ weights observed are most likely related to this decrease in body weight rather than a direct effect on the organs. Similarly, thyroid hormone assessments did not suggest a direct effect of atrazine on thyroid hormone homeostasis. It should be noted, however, that this report does not include information on the purity of the atrazine used in the study and that the histopathological data are not provided.

A.7.14 Tanaka, T., Kohno, H., Suzuki, R., Sugie, S. (2004) Lack of modifying effects of an estrogenic compound atrazine on 7,12

- dimethylbenz(a)anthracene

- induced ovarian carcinogenesis in rats. Cancer Lett. 210:129

- 137.

The effect of atrazine on DMBA

- induced ovarian c arcinogenesis was investigated in this study. The authors hypothesis is that the operative mode of action leading to ovarian adenocarcinomas involves hormonal disruption (particularly estrogen). Therefore, exposure to atrazine (which has been associated with hormone disruption) may affect ovarian carcinogenesis.

In this series of experiments, the researchers exposed 12

- 20 female Sprague

- Dawley rats to 0.5% DMBA (7,12

- dimethylbenz(a)anthracene) injected directly into the left ovaries. One week after ex posure to DMBA, animals were fed diets containing 0, 5, 50, or 500 ppm atrazine (purity unknown) for 50 weeks. Another group did not receive a DMBA injection but were exposed to 0 or 500 ppm atrazine through the diet. On week 51, the animals were sacrifi ced, necropsied and tissues with gross lesions, as well as, the liver, kidney, mammary gland, lung, and heart subjected to histopathological evaluation. In addition, immunohistochemistry evaluations for proliferating cell nuclear antigen (PCNA), expressio n of the androgen or estrogen receptors (α and β) and the BRCA

- 1 gene were conducted.

Neither DMBA, nor atrazine or DMBA + atrazine exposure led to any changes in ovarian, uterine, or body weight. DMBA exposure followed by dietary exposure to 500 ppm atr azine exposure led to a 10% statistically significant increase in absolute liver weight, consistent with what has been reported by other investigators. DMBA exposure led to a 45% incidence in ovarian adenocarcinomas vs 0% in control. Atrazine appeared to slightly modify the effect of DMBA since exposure to 5, 50, or 500 ppm atrazine subsequent to DMBA exposure resulted in a slight non

- statistically significant decrease in the incidence of ovarian tumors (22, 28, or 26%, respectively) compared to 45% incid ence after DMBA treatment

- alone. There were no compound

- related effectsPage 163 of 210 that could be unequivocally attributed to either DMBA, atrazine, or DMBA + atrazine exposure in the other tumor types seen in his study (malignant fibrous histiocytomas, hemangiomas, mammary tumors). PCNA

- labeling after treatment with DMBA alone was markedly increased ( ↑1644%) over unexposed animals. Atrazine exposure did not affect PCNA labeling index. Similarly, immunohistochemistry assessments did not reveal an impact of atrazin e exposure on the expression of the BRCA

- 1 protein or the estrogen or androgen receptors after DMBA treatment.

Reviewer’s Comments: The study authors concluded that atrazine did not result in any modifying effects on DMBA

- induced rat ovarian carcinogenes is. It should be noted, however, that though not statistically significant the incidence of ovarian tumors due to DMBA exposure was reduced by half when DMBA treatment was followed by atrazine exposure at dietary concentrations of 5

- 500 ppm (0.09

- 8.5 mg/r at/day). The toxicological significance of this finding, however, is unclear given the lack of dose

response over a two

- order of magnitude difference in atrazine exposures. However, when considered collectively, the data reported by Fujimoto, Son, and Tan aka and co

workers demonstrate that atrazine exposure did not accelerate tumor progression.

A.7.15 Ueda, M., Imai, T., Takizawa, T., Onodera, H., Mitsumori, K., Mtsui, T. and Hirose, M. (2005) Possible enhancing effects of atrazine on growth of 7,12

- dimethylbenz(a)anthracene

- induced mammary tumors in ovariectomized Sprague

- Dawley rats. Cancer Sci. 96(1):19

- 25.

The objective of this study was to evaluate the effect of atrazine exposure on late promotion/progression of mammary tumors in ovariectomized rats after treatment with 7,12

- dimethylbenz(a)anthracene (DMBA).

Two hundred and six female Sprague

- Dawley rats were used in the study. One hundred eighty six received a single gavage dose of DMBA (50 mg/kg) while 20 control rats received vehicle. Nin eteen weeks after DMBA treatment (when palpable tumor incidence was ≈ 50%), the animals were ovariectomized. The 89 rats bearing tumors at this time were designated as DMBA

- tumors (+) [Group A] while the 90 rats without tumors by week 19 were designated a s DMBA

- tumors (

- ) [Group B]. These rats were distributed among 4 subgroups (22

- 23 rats/group) that received 0, 5, 50, or 500 ppm atrazine (purity unknown) [approximately 0, 0.25, 2.5, and 25 mg/kg/day] through the diet for 34 weeks. A third group (Group C) consisting of animals not exposed to DMBA but exposed to atrazine at 0 or 500 ppm (10 rats/dose) for 34 weeks was also examined. Body weights, food consumption, the number of rats bearing tumors, number of tumors/rat, size of tumors, and location of pa lpable tumors were recorded once per week during atrazine

- treatment. At the terminal sacrifice, whole skin with mammary glands and tumors were collected. In addition, the liver, kidneys, uterus, pituitary glands, and adrenal glands were removed and weigh ed. Histopathology evaluations were conducted on mammary tumors, as well as, the liver, kidneys, spleen, lungs, heart, Zymbal glands, uterus, vagina, pituitary, adrenals, thyroids, mammary glands (not having gross indications of tumors), and scars of the ovariectomized regions. If indications that traces of ovarian tissue remained after the ovariectomy, thePage 164 of 210 animals were removed from the study. Thus the number of Group A animals treated with the various concentrations of atrazine was between 18

- 21/group a nd Group B animals treated with atrazine ranged between12

- 19 rats/group. Immunohistochemistry analysis was used to detect potential increases in proliferating

- cell nuclear antigen (PCNA), single strand DNA (ssDNA), and estrogen receptor α (ER α) in mammar y tumors (except for fibromas). Finally, estradiol, progesterone, prolactin, and luteinizing hormone levels were measured for Group A animals.

No effects on body weights or organ weights were reported for any treatment group. Mortality was higher in Gro up A (rats with early tumors) than Group B (rats with late tumors) but there was no apparent correlation to atrazine exposure.

Following ovariectomy, the number of Group A tumor

- bearing rats and total number of tumors palpated decreased by 6

- 29% and 50

- 6 8%, respectively, regardless of atrazine exposure. Similarly, the types of tumors ( i.e., benign vs . carcinoma) were not affected by atrazine treatment and though there was an apparent dose

- related increase in tumor size at doses ≥ 50 ppm (↑43

- 261%) there was no statistical significance due to the large variability in the Group A. Immunohistochemistry evaluations showed that Group A rats treated with 500 ppm atrazine showed an increase in the number of ERα

- positive tumor cells relative to control (3/3 vs. 2/5) as well as in the number of PCNA

- positive cells ( ↑92%, p<0.05) in malignant mammary tumors. The authors report that no changes in hormone levels that could be attributed to atrazine exposure were observed. However, no data were provided to corrobor ate this latter statement.

Interestingly, at the end of the study Group B rats ( i.e., DMBA

- treated rats with late tumors) had a statistically significant (p<0.05) increase in the number of tumor

- bearing rats exposed to 500 ppm atrazine relative to the Gro up B control animals (58% vs. 26% in control) yet a comparison with the Group A control animals revealed a lower incidence of tumor

- bearing rats in the atrazine

- treated animals than in the control. A ≈1300% increase in the size of tumors was also noted fo r the Group B rats treated with ≥ 50 ppm atrazine; again comparison to the Group A control animals revealed tha the tumors in the atrazine group were smaller than in the Group A controls. There was no evidence of a clear correlation between atrazine expos ure and the incidence of malignancies for either Group A or Group B rats.
Figure A

- 1 : Treatment GroupsPage 165 of 210 The authors concluded that atrazine has the potential to facilitate mammary tumors during the promotion/progression stage. These conclusions are based on increases in the Group B incidence of tumor

- bearing rats, number of carcinomas, tumor size at doses ≥ 50 ppm, and in the number of cells prol iferating (PCNA

- positive cells) and expressing ERα after exposure to 500 ppm atrazine.

The preponderance of the evidence, however, does not support the statement that atrazine contributes to mammary tumor development. For example, for Group A rats (tumor s present at week 19), there was no correlation between atrazine exposure and the number of tumor

- bearing rats, number of new tumors (appearing after ovariectomy), number of tumors/rat or the incidence of carcinomas. Moreover, the regression of palpable t umors observed after ovariectomy was similar for rats in the control and atrazine

- treated Group A rats. Although for Group B a statistically significantly higher incidence of tumor

- bearing rats is seen after exposure to 500 ppm atrazine relative to the Gr oup B control, it should be noted that the incidence in the Group B control is substantially lower than in the Group A control (Control B: 26% vs Control A: 72%). When the Group B 500 ppm atrazine

- treated rats are compared to the Group A controls, the inc idence of tumor

- bearing rats is lower after 500 ppm treatment than in the Group A controls. It should be noted that the incidence of tumor

- bearing rats in the Group B control was substantially lower than in the Group A control. When both control groups

a re combined (Group A + Group B), the incidence of tumor

- bearing rats at the Group B

500 ppm atrazine dose level is increased by 9% and is not statistically significant. 21 In fact, if Group B rats treated with 500 ppm atrazine are compared to the Group A co ntrol rats, the tumor incidence after atrazine exposure is lower than in the Group A controls.

For rats treated with 500 ppm atrazine in Group B (no tumors at week 19), the incidence of tumor

- bearing rats was lower than the number of tumor

- bearing rats in the Group A

21 Calculated by reviewer and analyzed using Fisher’s Exact test B

C A

Table excerpted from Ueda et al. , (2005) p. 22Page 166 of 210 control (58% vs 72%). Similarly, the number of carcinomas present in atrazine

- treated Group B rats did not show a dose response and at 500 ppm atrazine the carcinoma incidence was lower than for the Group A control rats.

With regards to the immunohistochemical evaluations, a statistically significant increase ( ↑92%, p< 0.05) in the number of PCNA

- positive cell carcinomas was noted for Group A rats treated with 500 ppm atrazine. This is not unexpected given that carcinomas are quickly proliferating lesions. Interestingly, atrazine exposure in and of itself does not appear to contribute to the increase in PCNA

- positive cells as evidenced by the fact that for late developing carcinomas (Group B rats) no statistically signifi cant increase was noted at the 500 ppm atrazine dose level. An increase in the incidence of ERα

- positive carcinomas was reported at the 500 pm atrazine level in Group A rats (100% vs 40% control). It should be noted, however, that a 100% incidence of ERα

- positive carcinomas was also reported for the control and 5 ppm atrazine Group B rats (late

developing tumors) while the high dose atrazine rats in Group B had an incidence of 75%.

Reviewer’s Comments: Based on the multiple lines of evidence presented in this study, a relationship between atrazine exposure and increases in the incidence of tumor rats cannot be drawn. In many instances it appears that the incidence reported for the 500 ppm atrazine Group B rats was comparable or at times lower than the incidence reported for the control Group A rats. Since control rats in Groups A and B were treated the same (only difference is detection of palpable tumors at week 19), a comparison of the treated groups with either control group or a combination of both is valid. While it is true that increases are occasionally seen in the 500 ppm atrazine

- treated rats that developed tumors after week 19 of the study when they were compared to the Group A control rats or when the Group A and Group B control rats (pre

- a nd post

- week 19 tumor development) were combined, the increase was no longer evident.

While there are a number of deficiencies in the conduct, interpretation, and reporting in the Ueda study, these data suggest that atrazine in an of itself does not indu ce mammary tumors in ovariectomized rats treated with a mammary tumor inducer thus demonstrating the importance of the ovarian hormones in the development of tumors in the mammary gland.Page 167 of 210 A.7.16 Yi, Kun Don, Breckenridge, Charles B, and Simpkins, James W. 2010. Cytotoxicity of Atrazine and its Metabolites in vitro. Unpublished data. Syngenta Crop Protection Inc.

In this study, the cytotoxicity of atrazine and its metabolites (diaminochlorotrizine [DACT], deisopropyl atrazine [DIA], ammeline [AM], hydr oxyatrazine [OH

- ATR]) was evaluated in four cell lines. The purity of the compounds was 97%. The four cell lines used were HT

- 22 cells (immortalized murine hippocampal neurons), MCF

- 7 cells (human breast adenocarcinomas), H295R cells (human adrenocortical carcinoma), and JEG03 cells (human placental choriocarcinoma). The cells were cultured in the appropriate media and the H295R, JEG

- 3, and MCF

- 7 cells were maintained at 100% confluency, while HT

- 22 cells were maintained at 50% confluency.

For the cell v iability assay, cells were seeded 24 hours before initiation at varying densities depending on the cell type in 96

- well plates: H295R cells

– 25,000 cells/well, JEG

- 3 cells

– 3500 cells/well, MCF

- 7 cells

– 15,000 cells/well, and HT

- 22 cells

- 3500 cells/wel l. Cells were exposed to atrazine or its metabolites at concentrations ranging from 0.001 μM to 300 μM in physiologically buffered saline (PBS) or in sterile water with dimethyl sulfoxide (DMSO) for up to 72 hours. After exposure, cell viability was

measur ed using the membrane

- permeant calcein

- AM dye. Calcein

- AM is a fluorogenic esterase substrate that is hydrolyzed to a fluorescent product in cells having esterase activity and intact membranes. Fluorescence was determined using an Infinity M120 microplate reader with an excitation/emission of 485/530 nm. The results, obtained in relative fluorescent units (RFU), were expressed as the percentage of untreated or vehicle treated control values. For each assay, six independent experiments were run with triplic ate wells for each treatment per experiment.

The effects of atrazine on mitochondrial activity was measured in H295R cells. H295R cells were seeded in 96

- well plates at 25,000 cells/well 24 hours before the initiation of the assay. Varying concentrations of atrazine were dissolved in DMSO (0.001

- 300 μM) or PBS (0.0003

- 300 μM) for 24 hours. Mitochondrial activity was assessed using a MTT assay.
Cytosolic reactive oxygen species (ROS) was estimated by measuring the amount of ROS by the fluorescent dye 2 ́,7  ́

- dichlorofluorescin diacetate (DCFH

- DA), which measures hydrogen peroxide (H 2 O 2 ). Cells were plated in 96

- well plates at a density of 25,000 cells/well 24 hours before the initiation of the assay. Cells were exposed to atrazine (10

- 300 μM) dissolved in D MSO or PBS. ROS production was monitored for up to 72 hours. DCF

- 2,7

- dichlorofluorescin fluorescence was determined at an excitation of 485 nm and an emission of 538 nm using an Infinity M120 microplate

reader.

The effects of atrazine on the cytotoxic effects of other known toxins was measured using H295R cells seeded in 96

- well plates at 25,000 cells/well 24 hours before the initiation of the assay. Varying concentrations of atrazine, DACT, DEA, DIA, ammeline, and OH

- ATR dissolved in DMSO were added. Cell viability was measured 24 hoursPage 168 of 210 after treatment. H295R cells were co

- treated with varying concentrations of atrazine and other know toxins (glutamate

– 15 mM; 3

- NPA

– 15 mM; H 2 O 2 – 250 μM) for 24 hours.

The results of this study showed that atrazine and its metabolites were not cytotoxic in four cell lines, irrespective of being solubilized in DMSO or PBS. Atrazine and its metabolites did not produce cytotoxicity as indicated by an absence of any effect on cell viability in two steroidogenic (H295R a nd JEG

- 3) and two non

- steroidogenic (HT

- 22 and MCF

- 7) cell lines. Atrazine had no effect on mitochondrial activity in H295R cells regardless of whether it was dissolved in DMSO or PBS. Atrazine did not induce oxidative stress in H295R cells, as measured b y cellular production of hydrogen peroxide (H 2 O 2 ).

The potential of atrazine or its metabolites to exacerbate the effects of other compounds known to induce cytotoxicity was evaluated. H295R cells were treated concurrently with atrazine and glutamate, or 3

- nitropropionic acid (3

- NPA), or H 2 O 2 24 hours. Atrazine and its metabolites did not affect the cytotoxicity of glutamate, 3

- NPA, or H 2 O 2.
Reviewer’s Comments: The methods section of the paper failed to include the evaluation of mitochondrial activity or measuring the effects of atrazine or its metabolites on cell viability when treated in conjunction with known cytotoxicants. Nevertheless, data from Yi et al. (2010) suggest that atrazine and its metabolites are not cytotoxic to two steroidogenic cell lines (H295R and JEG

- 3) as well as two non

steroidogenic cell lines (HT

- 22 and MCF

- 7).

A.8 INSULIN/MITOCHO NDRIA FUNCTION

Summary: Insulin/Mitochondria Function

In a recent publication in the electronic journal PLoS ONE, Lim et al. (2009) suggest that atrazine exposure leads to insulin resistance, fat accumulation, and increased weight gain by increasing blood glucose levels and lowe ring energy expenditure. The authors postulate that atrazine disrupts mitochondrial function by inhibiting mitochondrial complexes I, II and III, which interact with ubiquinone for electron transport. This hypothesis is based on atrazine’s known pesticid al mode of action by disrupting electron transport via plastoquinone which is similar in structure to ubiquinone in vertebrates. Mitochondria are involved in fat oxidation and insulin secretion pathways, so their disruption could cause steatosis or insuli n pathologies. However, mitochondrial inhibition should cause decreased insulin secretion, not increased as the authors propose. In fact, a recent publication in the Proceedings of the Academy of Sciences (PNAS) by Hoehn et al. (2009) demonstrated that

– contrary to the Lim et al. hypothesis

– mitochondrial inhibition reverses insulin resistance ( i.e. excess mitochondrial electron flow signals the over

- fed state to produce insulin resistance).

The authors state that atrazine exposure led to an increase in body weight and fat deposition regardless of the caloric intake. However, neither the body weight nor the fatPage 169 of 210 deposition increases ( ↑5

- 10%) reported in the study show a dose

- response. Moreover, the blood glucose levels reported by the authors after atra zine exposure though elevated relative to control are well within the normal range. Numerous deficiencies have been identified in this study and are described in detail in Appendix A. Amongst the most noteworthy deficiencies is the lack of several pieces of critical information in the material and methods, as well as inappropriate statistical analysis.

The authors concluded that exposure to atrazine at dose levels as low as 30 μg/kg/day resulted in significant mitochondrial dysfunction. Considering that NOAELs have been established in guideline in vivo toxicology studies at doses that are ≈ 60 to 300

- fold higher, this seems to be unlikely. Overall, the quality of this study was poor with numerous inconsistencies and deficiencies in the reporting thus re ndering it of limited utility in understanding the toxicological profile of atrazine.

In summary, Lim et al. are the only researchers that have proposed a relationship between atrazine exposure and mitochondrial dysfunction and obesity. The study used t o support this hypothesis is critically flawed. Moreover, the preponderance of the evidence indicates that atrazine exposure leads to decreases

– not **increases**

**–** in body weight. Consequently, the validity of these data is uncertain and renders it of litt le relevance for risk assessment purposes.

A.8.1 Lim, S., Ahn, S.Y., Song, I.C., Chung, M.H., Jang, H.C., Park, K.S., Lee, K

- U, Pak, Y.K., Lee, H.K. (2009). Chronic Exposure to the Herbicide, Atrazine, Causes Mitochondrial Dysfunction and Insulin Resista nce. PLoS ONE 4(4): e5186, p.1

- 11.

The authors have performed a series of in vivo and in vitro experiments to assess the extent to which low levels of atrazine in water effect mitochondrial function and insulin resistance. The authors have treated Spragu e

- Dawley rats with 0, 30 or 300 μg/kg/day atrazine in drinking water for 5 months. [These levels of atrazine are significantly lower than the dose levels used in most atrazine studies.] Rats were given either a standard diet or a high fat diet. The auth ors stated that there were no differences in water consumption during the study. The manuscript does not describe the vehicle or how often the water was prepared. In addition, the manuscript does not provide any information on the concentration of atrazi ne in the water or estimates of the water intake during the course of the study. In particular it is important to understand if the concentration of atrazine in the water or the vehicle changed the water consumption.

The following measurements were mad e as part of the in vivo experiments: body weight, visceral fat, motor activity, intramuscular and intrahepatic lipid content, plasma glucose, serum insulin, total cholesterol and triglycerides, intravenous glucose tolerance test, high insulin challenge ( hyperinsulinemic

- euglycemic study), energy expenditure, electron microscopy of mitochondria, and protein expression levels of mitochondrial OXPHOS complex. In vitro experiments were conducted to evaluate endogenous cellular oxygen consumption and mitochon drial respiratory chain activities. In general, the methods section lacks some important information and references to the methodsPage 170 of 210 used. For example, the methods state that motor activity was measured 4hrs/month. However, it is unclear whether this refer s to one 4 hr session or one hr/week. The authors performed student’s t

- tests and ANOVA. However, a 2

- way ANOVA with ATZ and diet as the independent variables may be more appropriate. Based on limited re

analysis conducted, further scrutiny of the stati stical approaches may be useful in the future.

Generally, the results section of this paper is hard to follow. Figure labels and legends are difficult to interpret.

Body weight and fat determinations. B ased on the results section, atrazine at these doses increased body weights in both normal and high fat diet, although, there is a flat dose response relationship. The lowest dose has an equivalent effect of the highest water concentration. Similarly, there is poor dose dependent effect on the fat

m easures, either the low dose is equivalent to the high dose or the low dose is more affected than the high dose (see intrahepatic lipid/water graph; Fig 1F). These results are in direct conflict with many other atrazine studies which suggest that atrazine results in decreased body weight, not increased.

ATZ and insulin and HOMA

- IR: The authors report a dose related increase in insulin and HOMA

- IR levels.

ATZ insulin resistance: Fasting plasma glucose levels of atrazine treated rats on the regular and high fat diets were higher than those in controls. The atrazine treated rats showed a reduced response to insulin loading, although the dose

- response curves are flat.

In vitro studies: The authors observe minor effects on mitochondrial function of atraz ine in their in vitro model at concentrations up to 460 uM. Rats given 150 mg/kg have peak blood concentrations of 7.5 uM. There seems to be a real disconnect between the in vitro concentrations needed to see effects on the mitochondria and the doses tha t produce effects on mitochondrial function in vivo . In order to achieve similar concentrations in vivo compared to the in vitro studies, exposures to atrazine would have to be well over 150 mg/kg.

Reviewer’s Comments: To believe their data, one would have to believe that atrazine has significant and near maximal effects on mitochondrial function at doses of 30 ug/kg/d (a LOEL) in animals on a normal diet. This seems unlikely given that most studies have not observed effects of atrazine until exposures are approximately 100

- 1000 times higher. Most notably, the results of this study are based on the statistical procedures used (which may be inappropriate). Preliminary analyses by Agency scientists do not confirm the results reported in the study. This study is not sufficiently robust for use in a regulatory context and should be repeated to confirm the findings.

A.9.1. PHARMACOKINET IC CALCULATIONS (a). Oral Gavage Dosing with Atrazine: Estimation of Plasma Half

- livesPage 171 of 210 Since atrazine was barely detected if at all by 24 hours post

- dosing, each treatment day was analyzed separately for elimination rate constant (kel) half

- life at dose levels of 3 and 10 mg/kg bw/day. At the high dose of 50 mg/kg bw/day, analysis was carried out on the la st treatment day (TD4) to include the washout period. In all cases, a significant linear behavior was observed. Figure 1a shows the plasma levels of atrazine plotted versus time in a semi

- logarithmic scale following each treatment day (TD) with 3 mg/kg bw/ day atrazine. Atrazine Analysis:

Figure 1a: Plasma elimination phase of atrazine resulting from oral gavage dosing with 3 mg/kg bw/day atrazine plotted versus time for treatment days 1

- 4 (TD1

- 4) on a semi

logarithmic scale. The dashed line represents the 95% confidence in terval of the linear regression.

Table 1a. Linear regression analysis for Figure 1a (3 mg/kg bw/day atrazine).

Table 1b: Summary of kel and half

- life estimates for atrazine at 3 mg/kg bw/day from Table 1a. ATRAZINE: 3 mg/kgbw/day: Elimination Phase Analysis

20 40 60 80 100

-8 -6 -4 -2 0 2 4 TD1 TD2 TD3 TD4

time (hr)Ln (plasma atrazine) (**μ**moles/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero?
F DFn, DFd
P value Deviation from zero?
Data Number of X values Maximum number of Y replicates
Total number of values Number of missing values Plasma ATRA (TD1, 3 mg/kg bw)

-0.9065 ± 0.06656
1.651 ± 0.1103
1.821
-1.103

-1.041 to -0.7724
1.429 to 1.873
1.623 to 2.051

0.8013
0.5696

185.5 1.000, 46.00
< 0.0001 Significant

48
1
48
102 Plasma ATRA (TD2, 3 mg/kg bw)

-1.175 ± 0.07359
30.23 ± 1.865
25.74
-0.8514

-1.325 to -1.024
26.41 to 34.05
25.56 to 25.93

0.9010
0.5534

254.7 1.000, 28.00
< 0.0001 Significant

30
1
30
120 Plasma ATRA (TD3, 3 mg/kg bw)

-0.8740 ± 0.04943
43.41 ± 2.476
49.67
-1.144

-0.9788 to -0.7692
38.16 to 48.66
49.42 to 49.92

0.9513
0.4293

312.7 1.000, 16.00
< 0.0001 Significant

18
1
18
132 Plasma ATRA(TD4, 3 mg/kg bw)

-0.8065 ± 0.08785
59.34 ± 6.517
73.57
-1.240

-0.9919 to -0.6212
45.59 to 73.09
73.04 to 74.04

0.8322
0.7809

84.30 1.000, 17.00
< 0.0001 Significant

19
1
19
131Page 172 of 210 Figure 2a: Plasma elimination phase of at razine resulting from oral gavage dosing with 10 mg/kg bw/day atrazine plotted versus time for treatment days 1

- 4 (TD1

- 4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression. logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression. kel (hr-1) Error
TD1 0.907 0.067
TD2 1.175 0.074
TD3 0.874 0.049
TD4 0.807 0.088
Mean = 0.941 0.069 t1/2 (hr)= 0.737 hr t1/2 (min)= 44.22 min

ATRAZINE:10 mg/kg bw/day elimination phase analysis

24 48 72 96

-8 -6 -4 -2 0 2 4 6 TD1 TD2 TD3 TD4

time (hr)Ln (plasma atrazine) (**μ**g/L)Page 173 of 210 Table 2a. Linear regression analysis for Figure 2a (10 mg/kg bw/day atrazine).

Table 2b: Summary of kel and half

- life estimates for atrazine at 10 mg/kg bw/day from Table 2a

.

Figure 3a: Plasma elimination phase of atrazine resulting from oral gavage dosing with 50 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression. Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values TD1

-0.5541 ± 0.03521 2.506 ± 0.1434 4.523 -1.805

-0.6246 to -0.4837 2.220 to 2.793 4.079 to 5.032

0.8077 0.8678

247.8 1.000, 59.00 < 0.0001 Significant

61 1 61 110 TD2

-0.6182 ± 0.04295 17.69 ± 1.142 28.61 -1.618

-0.7057 to -0.5306 15.36 to 20.01 28.12 to 29.19

0.8662 0.7523

207.2 1.000, 32.00 < 0.0001 Significant

34 1 34 137 TD3

-0.6041 ± 0.06079 31.66 ± 3.136 52.41 -1.655

-0.7305 to -0.4776 25.13 to 38.18 51.78 to 53.12

0.8246 0.8714

98.75 1.000, 21.00 < 0.0001 Significant

23 1 23 148 TD4

-0.5000 ± 0.04798 38.22 ± 3.645 76.44 -2.000

-0.5993 to -0.4007 30.68 to 45.76 75.68 to 77.25

0.8252 0.9193

108.6 1.000, 23.00 < 0.0001 Significant

25 1 25 146

10 mg/kg bw atrazine kel (hr-1) Error
TD1 0.554 0.035
TD2 0.618 0.043
TD3 0.604 0.061
TD4 0.500 0.048
Mean = 0.569 0.047 t1/2 (hr)= 1.22

ATRAZINE: 50 mg/kg bw/day TD4 elimination phase analysis

80 90 100 110 120

-10

-8 -6 -4 -2 0 2 4

time (hr)Ln (plasma atrazine,**μ**g/L)Page 174 of 210 Table 3a: Linear regression analysis for Figure 3a (50 mg/kg bw/day atrazine).

Table 3b: Summary of kel and half

- life estimates for atrazine at 50 mg/kg bw/day from Table 3a.

50 mg/kg bw atrazine kel (hr-1) Error
TD4 0.238 0.013 t1/2 (hr)= 2.91 Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values Plasma ATRA (50 mg/kg)

-0.2378 ± 0.01295 20.52 ± 1.041 86.31 -4.206

-0.2642 to -0.2114 18.40 to 22.65 85.20 to 87.59

0.9158 0.6354

337.1 1.000, 31.00 < 0.0001 Significant

33 1 33 27Page 175 of 210 DEA Analysis:

Figure 4a: Plasma elimination phase of DEA resulting from oral gavage dosing with 3 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 4a: Linear regression analysis for DEA (Figure 4a, 3 mg/kg bw/day atrazine). DEA: 3 mg/kg bw/day atrazine-elimination phase analysis

75 80 85 90 95 100

-2 0 2 4 6

time (hr)Ln (plasma DEA) (**μ**g/L)

DEA: 10 mg/kg bw atrazine- elimination phase analysis

80 100 120 140 160 180 200

-10

-5 0 5 10

time (min)Ln (plasma DEA) (**μ**g/L) Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DEA ( **μ** g/L, 3mg/kg ATRA)

-0.2040 ± 0.009172 18.23 ± 0.7376 89.34 -4.902

-0.2227 to -0.1853 16.72 to 19.73 88.29 to 90.55

0.9392 0.4239

494.6 1.000, 32.00 < 0.0001 Significant

34 1 34 2Page 176 of 210 Figure 5a: Plasma elimination phase of DEA resulting from oral gavage dosing with 10 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 5a: Linear regression analysis for DEA (Figure 4a, 10 mg/kg bw/day atrazine).

Figure 6a: Plasma elimination phase of DEA resulting from oral gavage dosing with 50 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 6a: Linear regression analysis for DEA (Figure 6 a, 50 mg/kg bw/day atrazine). Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DEA

-0.09281 ± 0.003397 10.37 ± 0.3795 111.8 -10.78

-0.09960 to -0.08602 9.613 to 11.13 109.4 to 114.2

0.9222 0.8949

746.5 1.000, 63.00 < 0.0001 Significant

65 1 65 11

DEA: 50 mg/kg bw atrazine- elimination phase analysis

80 100 120 140 160 180 200

-4 -2 0 2 4 6 8

time (hr)Ln (plasma,**μ**g/L)Page 177 of 210 DIA Analysis A small, secondary (β) phase of elimination was sometimes observed for this metabolite at the later time points, but given its magnitude compared to the much greater α

- phase, it was neglected.

Figure 7a: Plas ma elimination phase of DIA resulting from oral gavage dosing with 3 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 7a : Linear regression analysis for DIA (Figure 7a, 3 mg/kg bw/day atrazine). Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DEA

-0.06783 ± 0.003481 9.720 ± 0.4158 143.3 -14.74

-0.07477 to -0.06088 8.891 to 10.55 138.8 to 148.5

0.8369 1.103

379.8 1.000, 74.00 < 0.0001 Significant

76 1 76 0

DIA: 3 mg/kg bw atrazine - analysis of elimination phase (from 72 - 80 hrs)

72 74 76 78 80 0 2 4 6 8

time (hr)Ln (plasma DIA) (**μ**g/L)Page 178 of 210 Figure 8a: Plasma elimination phase of DIA resulting from oral gavage dosing with 10 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 8a: Linear regression analysis for DIA (Figure 8a, 10 mg/kg bw/day atrazine). Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DIA (3mg/kg)

-0.5636 ± 0.03036 45.99 ± 2.290 81.60 -1.774

-0.6266 to -0.5006 41.24 to 50.74 80.90 to 82.46

0.9400 0.4442

344.7 1.000, 22.00 < 0.0001 Significant

24 1 24 0

DIA: 10 mg/kg bw atrazine- elimination phase analysis

80 90 100 110 120

-4 -2 0 2 4 6 8 10

time (hr)Ln (plasma DIA) (**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DIA, 10 mg/kg bw ATRA

-0.2425 ± 0.01456 22.99 ± 1.219 94.83 -4.124

-0.2720 to -0.2129 20.52 to 25.47 92.98 to 97.04

0.8852 1.023

277.5 1.000, 36.00 < 0.0001 Significant

38 1 38 4Page 179 of 210 Figure 9a: Plasma elimination phase of DIA resulting from oral gavage dosing with 50 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 9a: Linear regression analysis for DIA (Figure 9 a, 50 mg/kg bw/day atrazine). DIA: 50 mg/kg bw atrazine- elimination phase analysis

80 90 100 110 120 130 140

-4 -2 0 2 4 6 8 10

time (hr)Ln (plasma DIA) (**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DIA

-0.1757 ± 0.006532 19.72 ± 0.5857 112.2 -5.692

-0.1889 to -0.1625 18.53 to 20.90 110.2 to 114.5

0.9439 0.7099

723.6 1.000, 43.00 < 0.0001 Significant

45 1 45 3Page 180 of 210 DACT

Figure 10a: Plasma elimination phase of DACT resulting from oral gavage dosing with 3 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 9 5% confidence interval of the linear regression. Table 10a: Linear regression analysis for DACT (Figure 10a, 3 mg/kg bw/day atrazine). DACT: 3 mg/kg bw atrazine- elimination phase analysis

80 100 120 140 160 180 200

-8 -4 0 4 8

time (hr)Ln(Plasma,**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DACT (3 mg/kg bw ATRA)

-0.08730 ± 0.006230 12.80 ± 0.7536 146.7 -11.45

-0.09974 to -0.07486 11.30 to 14.31 140.0 to 154.7

0.7400 2.064

196.3 1.000, 69.00 < 0.0001 Significant

71 1 71 11Page 181 of 210 Figure 11a: Plasma elimination phase of DACT resulting from oral gavage dosing with 10 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression.

Table 11a: Linear regression analysis for DACT (Figure 10a, 10mg/kg bw/day atrazine). DACT: 10 mg/kg bw atrazine- elimination phase analysis

72 96 120 144 168 192

-10

-5 0 5 10

time (hr)Ln (plasma DACT,**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DACT (ug/L)

-0.08449 ± 0.006908 14.05 ± 0.9067 166.3 -11.84

-0.09830 to -0.07068 12.24 to 15.87 158.2 to 176.8

0.7070 2.100

149.6 1.000, 62.00 < 0.0001 Significant

64 1 64 6Page 182 of 210 Figure 12a: Plasma elimination phase of DACT resulting from oral gavage dosing with 50 mg/kg bw/day atrazine plotted versus time for treatment day 4 (TD4) on a semi

- logarithmic scale. The dashed line represents the 95% confidence interval of the linear regression

. Table 11a: Linear regression analysis for DACT (Figure 10a, 50 mg/kg bw/day atrazine).

(b). Oral Gavage Dosing with Atrazine: Estimation of Plasma AUC Plasma AUC was estimated based on the trapezoid rule as implemented by Graphpad Prism (version 5). In the case of atrazine, DEA, and DIA, plasma levels at the last time point of 24 hours were very minimal, and thus no correction was made for estimating the area from the last time point to infinity. Only in the case of DACT, a correction was made by esti mating the area from the last time point to infinity by dividing the last plasma time point by the estimated elimination rate constant. DACT: 50 mg/kg bw atrazine- elimination phase analysis

84 96 108 120 132 144 156 0 2 4 6 8 10

time (hr)Ln (plasma DACT,**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values -0.07522 ± 0.01020 14.69 ± 1.186 195.3 -13.29

-0.09596 to -0.05448 12.28 to 17.11 177.3 to 226.7

0.6154 1.196

54.41 1.000, 34.00 < 0.0001 Significant

36 1 36 0Page 183 of 210 Figure 13a: 24

- h plasma profile for atrazine following oral gavage dosing with 3, 10, and 50 mg/kg bw atrazine.

Table 13a: AUC estimates for plasma levels of atrazine from Figure 13a. 0 1 2 3 4 5 6 7 8 0 10 20 30 40

10 12 14 16 18 20 22 24 3 mg/kg bw/day ATRA 10 mg/kg bw/day ATRA 50 mg/kg bw ATRA

time (hr)Plasma atrazine (**μ**g/L)

Baseline Total Area Total Peak Area Number of Peaks

Peak 1 First X= Last X= Peak X= Peak Y= Area= %Area= 3 mg/kg bw/day ATRA 0.0 6.411 6.361 1.000

0.0 6.000 0.3333 6.432 6.361 100.0 10 mg/kg bw/day ATRA 0.0 24.87 24.72 1.000

0.0 18.00 0.3333 18.04 24.72 100.0 50 mg/kg bw ATRA 0.0 109.6 109.6 1.000

0.0 24.00 0.1667 31.07 109.6 100.0

3 mg/kg bw atrazine

0 1 2 3 4 5 6 0 50 100 150 200 250 300

8 10 12 14 16 18 20 22 24 DEA DIA

time (hr)Plasma Levels (**μ**moles/L)Page 184 of 210 Figure 14a: 24

- h plasma profile for DEA and DIA following oral gavage dosing with 3 mg/kg bw atrazine. Table 14a: AUC estimates for plasma levels of DEA and DIA from Figure 14a.

Baseline Total Area Total Peak Area Number of Peaks

Peak 1 First X= Last X= Peak X= Peak Y= Area= %Area= DEA (ug/L) 0.0 153.5 153.5 1.000

0.0 24.00 0.3333 58.17 153.5 100.0 DIA (ug/L) 0.0 370.3 370.3 1.000

0.0 24.00 0.3333 220.7 370.3 100.0Page 185 of 210 Figure 15a: 24

- h plasma profile for DACT following oral gavage dosing with 3 mg/kg bw atrazine

Table 15a: AUC estimates for plasma levels of DACT from Figure 15a. DACT AUC estimate - 3 mg/kg bw atrazine

0 2 4 6 8 10 12 14 16 18 20 22 24 0 200 400 600 800 1000

time (hr)Plasma Levels (**μ**g/L)

Baseline Total Area Total Peak Area Number of Peaks

Peak 1 First X= Last X= Peak X= Peak Y= Area= %Area= DACT 0.0 7947 7947 1.000

0.0 24.00 2.000 682.0 7947 100.0

**∞** AUC
0 last AUC
0 **∞** AUC last = +

= 7947 + Cplast
kel

= 7947 + 84 mg/L
0.0873/hr

= 7947 + 962.2

= 8909.2 (mg/L)*hrPage 186 of 210 Figure 16a: 24

- h plasma profile for DEA, DIA, and DACT following oral gavage dosing with 10 mg/kg bw atrazine Table 16a: AUC estimates for plasma levels of DEA, DIA, and DACT from Figure 16a. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0 500 1000 1500 2000 2500 DEA DIA DACT

time (hr)Plasma Levels (**μ**g/L)

Baseline Total Area Total Peak Area Number of Peaks

Peak 1 First X= Last X= Peak X= Peak Y= Area= %Area= DEA 0.0 465.3 465.3 1.000

0.0 24.00 1.000 141.0 465.3 100.0 DIA 0.0 1336 1336 1.000

0.0 24.00 1.000 614.2 1336 100.0 DACT 0.0 25139 25139 1.000

0.0 24.00 6.000 2125 25139 100.0

**∞** AUC
0 last AUC
0 **∞** AUC last = +

= 25139 + Cplast
kel

= 25139 + 367.83 mg/L
0.0845/hr

= 25139 +

= 29492.02 (mg/L)*hr DACT:

4353.02Page 187 of 210 Figure 17a: 24

- h plasma profile for DEA, DIA, and DACT following oral gavage dosing with 10 mg/kg bw atrazine 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0 2000 4000 6000 8000 10000 DEA DIA DACT

time (hr)Plasma Levels (**μ**g/L)Page 188 of 210 Table 17a: AUC estimates for pl asma levels of DEA, DIA, and DACT from Figure 17a.

Baseline Total Area Total Peak Area Number of Peaks

Peak 1 First X= Last X= Peak X= Peak Y= Area= %Area= DEA 0.0 2277 2277 1.000

0.08333 24.00 0.7500 219.7 2277 100.0 DIA 0.0 7255 7255 1.000

0.08333 24.00 2.000 1066 7255 100.0 DACT 0.0 121819 121819 1.000

0.08333 24.00 8.000 8614 121819 100.0

**∞** AUC
0 last AUC
0 **∞** AUC last = +

= 121819 + Cplast
kel

= 121819 + 2053.4 mg/L
0.0752/hr

= 121819 +

= 149124.9 (mg/L)*hr DACT:

27305.9Page 189 of 210 (b). Dietary Dosing with Atrazine: Estimation of Plasma Half

- lives

Figure 18a: Elimination phase analysis of DEA following dosing with atrazine through the diet at the nominal dose level of 3 mg/k g bw/day atrazine. DEA: 3 mg/kg/day atrazine: elimination phase analysis

96 120

-6 -4 -2 0 2 4

time (hr)Ln (plasma DEA) (**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DEA (3 mg/kg bw atrazine)

-0.1871 ± 0.01258 19.90 ± 1.365 106.4 -5.344

-0.2126 to -0.1616 17.14 to 22.67 105.2 to 107.4

0.8534 0.6294

221.2 1.000, 38.00 < 0.0001 Significant

40 1 40 1Page 190 of 210 Figure 19a: Elimination phase analysis of DEA following dosing with atrazine through the diet at the nominal dose level of 10 mg/kg bw/day (actual dose level = 9 mg/kg bw/day). DEA: 9 mg/kg/day atrazine - elimination phase analysis

96 120 144 168 192

-6 -4 -2 0 2 4

time (hr)Ln (plasma DEA) (**μ**g/L)

Best-fit values

Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DEA (10 mg/kg atrazine)

-0.07614 ± 0.003619 9.377 ± 0.4506 123.2 -13.13

-0.08338 to -0.06890 8.476 to 10.28 120.9 to 125.4

0.8824 0.6570

442.6 1.000, 59.00 < 0.0001 Significant

61 1 61 17Page 191 of 21 0 Figure 20a: Elimination phase analysis of DEA following dosing with atrazine through the diet at the nominal dose level of 50 mg/kg bw/day (actual dose level = 43 mg/kg bw/day). DEA: 43 mg/kg/day atrazine - elimination phase analysis

96 120 144

-2 0 2 4 6

time (hr)Ln (plasma DEA) (**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DEA (43 mg/kg bw/day atrazine)

-0.1042 ± 0.004897 14.15 ± 0.5488 135.8 -9.598

-0.1141 to -0.09432 13.04 to 15.26 133.5 to 138.6

0.9078 0.3849

452.7 1.000, 46.00 < 0.0001 Significant

48 1 48 0Page 192 of 210 Figure 21a: Elimination phase analysis of DIA following dosing with atrazine through the diet at the nominal dose level of 3 mg/kg b w/day. DIA: 3 mg/kg bw/day atrazine - elimination phase analysis

96 120

-4 -2 0 2 4

time (hr)ln (plasma levels,**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DIA ( **μ** g/L)

-0.2309 ± 0.01586 24.41 ± 1.693 105.7 -4.331

-0.2632 to -0.1986 20.96 to 27.86 104.7 to 106.7

0.8688 0.6730

211.9 1.000, 32.00 < 0.0001 Significant

34 1 34 7Page 193 of 210 Figure 22a: Elimination phase analysis of DIA following dosing with atrazine through the diet at the nominal dose level of 10 mg/kg bw/day (actual dose level = 9 mg/kg bw/day). DIA: 9 mg/kg bw/day - elimination phase analysis

100 120

-4 -2 0 2 4 6

time (hr)ln (plasma levels,**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values -0.2485 ± 0.01497 27.08 ± 1.612 109.0 -4.025

-0.2788 to -0.2181 23.82 to 30.35 108.1 to 110.0

0.8816 0.7126

275.4 1.000, 37.00 < 0.0001 Significant

39 1 39 3Page 194 of 210 Figure 23a: Elimination phase analysis of DIA following dosing with atrazine through the diet at the nominal dose level of 50 mg/kg bw/day (actual dose level = 43 mg/kg bw/day). DIA: 43 mg/kg bw/day - elimination phase analysis

96 108 120 132

-2 0 2 4 6 8

time (hr)Ln (Plasma) (**μ**g/L)

Best-fit values

Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DIA ( **μ** g/L)

-0.2428 ± 0.01319 27.99 ± 1.433 115.3 -4.119

-0.2694 to -0.2161 25.09 to 30.88 114.2 to 116.5

0.8943 0.6867

338.6 1.000, 40.00 < 0.0001 Significant

42 1 42 0Page 195 of 210 Figure 24a: Elimination phase analysis of DACT following dosing with atrazine through the diet at the nominal dose level of 3 mg/kg bw/day. DACT: 3 mg/kg bw atrazine- elimination phase analysis

96 120 144 168 192

-4 -2 0 2 4 6 8

time (hr)Plasma DACT (ug/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values Plasma DACT (3 mg/kg bw atraz)

-0.08977 ± 0.001865 14.68 ± 0.2404 163.5 -11.14

-0.09350 to -0.08605 14.20 to 15.16 161.7 to 165.5

0.9735 0.4019

2316 1.000, 63.00 < 0.0001 Significant

65 1 65 12Page 196 of 210 Fi gure 25a: Elimination phase analysis of DACT following dosing with atrazine through the diet at the nominal dose level of 10 mg/kg bw/day (actual dose level = 9 mg/kg bw/day). DACT: 9 mg/kg bw/day atrazine- elimination phase analysis

96 120 144 168 192 216 240
-2 0 2 4 6 8

time (hr)Plasma Levels (**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DACT (9 mg/kg bw atrazine)

-0.07287 ± 0.002136 14.02 ± 0.2814 192.4 -13.72

-0.07714 to -0.06860 13.46 to 14.58 188.6 to 196.7

0.9440 0.5050

1164 1.000, 69.00 < 0.0001 Significant

71 1 71 7Page 197 of 210 Figure 26a: Elimination phase analysis of DACT following dosing with atrazine through the diet at the nominal dose level of 50 mg/kg bw/day (actual dose level = 43 mg/kg bw/day). DACT: 43 mg/kg bw/day atrazine - elimination phase analysis

96 120 144 168 192 0 2 4 6 8 10

time (hr)Ln (plasma levels,**μ**g/L)

Best-fit values Slope Y-intercept when X=0.0 X-intercept when Y=0.0 1/slope 95% Confidence Intervals Slope Y-intercept when X=0.0 X-intercept when Y=0.0 Goodness of Fit R square Sy.x Is slope significantly non-zero? F DFn, DFd P value Deviation from zero? Data Number of X values Maximum number of Y replicates Total number of values Number of missing values DACT (43 mg/kg bw atrazine)

-0.07298 ± 0.001597 15.74 ± 0.2190 215.7 -13.70

-0.07617 to -0.06979 15.31 to 16.18 212.1 to 219.7

0.9649 0.4387

2088 1.000, 76.00 < 0.0001 Significant

78 1 78 0Page 198 of 210 (III). Estimating the volume of distribution at steady state for plasma triazines

Figure 27a: First moment

- time curve following dosing of rats with 100 mg/kg bw atrazine

100 mg/kg Atrazine: Area under the first moment curve (AUMC) analysis

0 100 200 300 400 0 100 200 300 400 500

Paul et al. 1993

time (hr)Cp*t (mg*hr/L)

Baseline Total Area Total Peak Area Number of Peaks

Peak 1 First X= Last X= Peak X= Peak Y= Area= %Area= AUMC 0.0 57445 57445 1.000

0.0 336.0 24.00 386.9 57445 100.0Page 199 of 210 Figure 28a. Area under the first moment curve analysis for estimating Vdss.

A.9.2. PHARMACOKINETIC STUDIES

- MINIREVIEWS

A Physiologically Based Pharmacokinetic (PBPK) Model for Atrazine and its Main Metabolites in the Adult Male C57BL/6 Mouse Toxicology and Applied Pharmacology 251 (2011) 16

- 31

This manuscript describes the development of a PBPK model in the adult male C57BL/6 mouse based on a previously published disposition study of atrazine (ATRA) and its chlorinated metabolites deethylatrazine (DEA), deisopropylatrazine (DIA), and diaminochlo rotriazine (DACT) following a single oral gavage dose of 5, 25, 125, and 250 mg/kg bw of ATRA (Ross et al. 2009). The PBPK model consists of four submodels describing the pharmacokinetics of each species (ATRA, DEA, DIA, and DACT) linked via systemic circu lation into an integrated model. In the case of ATRA and its main 0 **∞** Vdss = 0 **∞** Dose *

Where AUCM is the area under the first moment of the plasma concentration-time curve and AUC is the area under the concentration-time curve AUCM (mg*hr 2 /L)

AUC 2 (mg 2 *hr 2 /L 2 )

For 100 mg/kg atrazine, AUCM to last = 57445 mg*hr 2 /L

0 **∞**

AUCM = 57445 + Cp last *t last kel + Cp last
kel 2

= 57445 + 50.3 + 0.15
0.013 hr -1 (0.013 hr -1 ) 2

= 62201.83 mg*hr 2 /L

For 100 mg/kg atrazine, AUC to last = 963 (mg/L)*hr

0 **∞**

AUC = 963 + 0.148/0.014 = 974.4 (mg/L)*hr

Vdss = 100 mg/kg bw * (974.4mg*hr/L) 2 = 6.55 L/kg bw 62201.83 mg*hr 2 /LPage 200 of 210 metabolite DACT, the compartments include blood, brain (potential target organ), liver (primary site of metabolism), kidney (primary site of elimination), rapidly

- and slowly

perfused compar tments. The submodels for DEA and DIA consist of five compartments, namely blood, brain, liver, slowly

- and rapidly

- perfused compartments. The uptake of atrazine from the gut was described using a 2

- compartment model used previously for lipophilic chemical s consisting of an initial rapid absorption phase from the stomach lumen, followed by gastric emptying into the intestinal lumen where a slow, sustained uptake takes place into the systemic circulation. All three rate constants were estimated via visually fitting plasma concentration of ATRA and its chlorinated metabolites. The following assumptions were used in modeling of ATRA and its metabolites: flow

- limited distribution, saturable metabolism, and first

- order urinary elimination. Binding to plasma prote ins and red blood cells was included in the model via second order equations assuming that 26% of ATRA and DACT are bound to plasma proteins and 1.5% of total chlorinated triazines are bound to RBCs based on information from previous studies. The model was calibrated with dosimetry data from the highest dose tested of 250 mg/kg bw (Ross et al. 2009). Partition coefficients (PCs) were also estimated from the highest dose tested based on the area under the curve (AUC) ratio of the particular tissue over that of plasma. Due to the lack of appropriate information, liver PCs for DEA and DIA were estimated as the 4

- h time point concentration in the liver over that of plasma. In vivo hepatic metabolism of ATRA to DEA and DIA was estimated by manually adjusting in v itro metabolic rates in the mouse for better fit to the plasma data. Optimal fitting was obtained when in vitro metabolic rates were increased by 2

- fold. Metabolism of DEA and DIA to DACT was based on the rat metabolic rate used in a previous rat model (Mc Mullin 2007)

This modeling effort in the mouse involved significant parameter

- fitting to the measured data of mice dosed with 250 mg/kg bw ATRA. Significant parameter adjustments were made to oral uptake, metabolism, and urinary excretion. Being that dose dependent changes were observed in the oral uptake of ATRA as well as PCs, questions remain regarding whether the data for the highest dose of 250 mg/kg is appropriate for model calibration. While plasma profile of DACT can be reasonably described by the model at the high dose levels of 250, 125, and 25 mg/kg ATRA, plasma levels of ATRA, DEA, and DIA were not, particularly at the later time point of 24 hours. Brain levels of ATRA and DACT were much better simulated than liver levels were at the same high d ose levels. Simulation of brain levels of ATRA and DACT improved but still were not adequately described when PCs in the model were replaced with those based on the low dose level of 5 mg/kg ATRA, indicating potentially dose

- dependent changes in PCs. The m odel should have been calibrated with data from a lower dose of ATRA to be more relevant to risk assessment applications. When the mouse PBPK model was scaled to rats, the plasma profile of ATRA was significantly over

- predicted while doing a better job sim ulating plasma levels of DACT. This species extrapolation exercise underscores the limited potential of this current model for use in cross

- species dosimetry extrapolation, though it represents a good effort in identifying data needs for a more reliable mo del.Page 201 of 210 **In Vitro** Metabolism of Atrazine in Rat and Human Hepatocytes

Jennifer A. Thomas, Ph.D., WIL Research Laboratories, LLC (Report # WIL

- 639084), Sponsored by Syngenta Crop Protection

The in vitro metabolism of atrazine (ATZ) to its chlorometabo lites, deisopropylatrazine (DIA), deethylatrazine (DEA), and diaminochlorotriazine (DACT) was investigated using cryopreserved hepatocytes from rat and human donors during a 4

- hour period. The initial concentrations of atrazine were 1.7, 1.0, or 0.5 μM. Sa mples were collected after 0, 5, 10, 20, 30, 45, 90, 120, 180, and 240 minutes of incubation at which time they were snap

- frozen and sent for analysis of ATZ, DIA, DEA, and DACT. The hepatocyte suspension cultures were deemed acceptable for investigat ing the in vitro metabolism of atrazine based on confirmation of enzymatic activity and cell viability. The de

- ethylation of 7

- ethoxycoumarin was used as confirmation of enzymatic activity. The starting hepatocyte viability was > 70% for both rats and huma ns as required by the protocol but decreased to final average cell viability of 29% in rats and 49% in humans. The rates of metabolism, however, were not affected by decreases in viability and/or hepatocyte enzyme activity. Atrazine was metabolized wi thin 30 to 60 minutes concurrent with the appearance of DEA and DIA. After approximately 30 to 45 minutes of incubation, the concentrations of DEA and DIA began declining concomitantly with the appearance of DACT progressively over the entire 4 hour incuba tion period. Rat hepatocytes metabolized atrazine preferentially to DIA, whereas human hepatocytes preferentially metabolized ATZ to DEA. Atrazine fell to non

- detectable levels after 30 to 60 minutes, depending upon the initial concentration. The time to m aximum levels (tmax) for DEA, DIA, and DACT was 20, 30, and 240 minutes in rat hepatocytes exposed to 0.5 μM atrazine, whereas with human hepatocytes, the corresponding times were 30, 30, and 180 minutes, respectively. The experimental maximum levels (Cmax ) for DEA, DIA, and DACT were 14, 36, and 27 ng/mL for rat hepatocytes, and the corresponding Cmax values for human hepatocytes were 50, 11, and 27 ng/mL, respectively. When adjusted for decreases in cell viability, the corresponding Cmax were 21, 58, and 127 ng/mL for rat hepatocytes and 80, 18, and 63 ng/mL, respectively, for human hepatocytes. The metabolism rates obtained from this in vitro study can be used to parameterize a PBPK model for atrazine and its chlorinated metabolites DEA, DIA, and DACT.

P BPK Model for Atrazine and its Chlorinated Metabolites Jerry Campbell, Melvin Andersen, and Harvey Clewell, the Hamner Institutes for Health Sciences, Center for Human Health Assessment, Sponsored by Syngenta Crop Protection Although the Agency has not performed a thorough review and evaluation of the model, a preliminary review indicates that the original McMullin 2007 model (McMullin et al. 2007) has been expanded to include compartments of suspected target tissues including mammary gland, hypothal amus, pituitary, testes/ovaries, and adrenals. The oral uptake of atrazine has also been refined by removing a saturable uptake description and replacing it with separate compartments for insoluble/bound and free fractions of atrazine as well as describing the potential slow release of bound atrazine.Page 202 of 210 New empirically determined tissue:blood partition coefficients and hepatic in vitro metabolic rates were also incorporated. Most importantly, however, was the model calibration performed with the new in vivo t ime

- intensive rat pharmacokinetic study described above where the chlorinated metabolites DEA, DIA, and DACT were individually monitored in female SD rats following dosing with atrazine via the oral route of administration (Coder 2011). The Agency will con sider using the newly improved PBPK in the future when it has undergone a thorough review and evaluation process.

A. 10 MIXTURES

A.10.1 Enoch, R.R., Stanko, J.P., Greiner, S.N., Youngblood, G.L. Rayner, J.L., Fenton, S.E., 2007. Mammary Gland Develop ment as a Sensitive End Point after Acute Prenatal Exposure to an Atrazine Metabolite Mixture in Female Long

- Evans Rats. Environmental Health Perspectives. 115(4): 541

- 547. Stanko, J., Enoch, R., Rayner, J., Davis, C., Wolf, D., Malarkey, D., and Fenton S. 2010. Effects of Prenatal Exposure to a Low Dose Atrazine Metabolite Mixture on Pubertal Timing and Prostate Development of Male Long Evans Rats. Reproductive Toxicology. 30:540

- 549.

Two articles, Enoch et al. 2007 and Stanko et al . 2010, were produce d from one study. The Enoch et al article focused on mammary gland development and Stanko et al. 2010 focused on reproductive development of male rats following prenatal exposure to an atrazine metabolite mixture (AMM).

The study was conducted in 2 repli cate blocks with 5 treatment groups/block. Based on the results from these blocks, a third block was conducted to include a 180

- day necropsy. In this study, the investigators dosed pregnant female Long

- Evans rats (n ≥6 treatment/block) with atrazine or a m ixture of atrazine and its metabolites from gestational day (GD) 15

- 19. Exposure was conducted on GD 15

- 19 because this dosing window is a critical period of development for both the mammary gland and prostate. The atrazine metabolite mixture (AMM) was ma de up of atrazine, DACT, DEA, DIA, and hydroxyatrazine (HA) at 0, 0.09 mg/kg AMM, 0.87 mg/kg AMM, 8.73 mg/kg AMM, and 100 mg/kg atrazine (positive control). The 100 mg/kg atrazine group was used as a positive control for effects on mammary gland developme nt. Several endpoints were measured but the focus of the Enoch et al. 2007 article is on development of the mammary gland following in utero exposure. The animals in the Stanko et al. 2010 article were the siblings of those in Enoch et al. 2007 and focuse d on pubertal timing and prostate development following in utero exposure. Reproductive organs were weighed and hormone measurements were taken.

Enoch **et al.** 2007 : Two female pups/dam (for ≥ 5 dams) were necropsied on post

- natal days (PND) 4, 25, 33, 4 0, and 60. For mammary gland developmental evaluations, the 4 th and/or 5 th inguinal mammary gland were whole mounted and examined subjectivelyPage 203 of 210 using a 1

- 4 scoring system (1 = poor development/structure; 4 = normal development/structure). The scoring criteria varied depending on the age of the pups. The scoring system is intended to evaluate: primary, secondary, lateral branching; longitudinal growth toward the lymph node; budding patterns (or lack thereof); and the presence of terminal end buds or ducts.

Pup body weights were increased compared to controls in the three AMM treated groups. Dam weights were not affected; the two lower doses o f AMM showed 7

- 12% increases in body weight compared to controls. The day of vaginal opening was the same in treated groups compared to controls. Similarly, the estrous cycles in treated animals were similar to controls. The AMM treated animals exhibite d increased pituitary weights compared to controls.

With respect to the mammary gland development findings, the investigators evaluated pups at PND 4, at weaning, PND 33, PND 40, and PND 60. The results are provided below. Representative images are pr ovided in Figure 5 of the paper. The authors argue that adverse findings are observed as low as 0.09 mg/kg AMM. There is, however, a weak dose

- response curve. In fact, increasing the AMM dose by 100

- fold changes the response very little.

Stanko et a l. 2010: Two male pups/dam from ≥5 dams were necropsied on PND 120 in blocks one and two (n ≥10) and on PND 120 and 180 in block three. For prostate evaluations, the ventral and lateral lobes of the prostate were processed by routine methods and embedded i n paraffin block. The lobes were graded as a whole because the inflammation involved both the ventral prostate (VP) and lateral prostate (LP). Severity of inflammation was scored as follows: “1

- minimal multifocal interstitial infiltration of inflammator y cells involving less than 10% of the gland, 2

– multifocal to coalescing moderate interstitial inflammation affecting 10

- 25% of the gland, with or without focally marked accumulations of perivascular collections of inflammatory cells or mild fibrosis, 3

– multifocal, marked interstitial inflammation involving 25

- 75% of the gland, with or without focally marked accumulations of perivascular collections of inflammatory cells, moderate fibrosis, or sloughing and necrosis of the epithelial lining with intralu minal accumulations of neutrophils (micro

- abscesses), or 4

- diffuse and extensive interstitial inflammation involving > 75% of the gland often with interstitial fibrosis and occasional microabscesses.”Page 204 of 210 No effect was seen on pup body weight of the AMM or A TZ treated groups compared to controls on PND 4 or 21. A slight statistically significant decrease in pup body weight was seen in the 0.09 mg AMM group (5.7%) on PND 120, but this is not considered to be biologically relevant. An 8

- 9% increase in pup body weight compared to controls was seen in the 0.87 and 8.73 AMM dose groups, respectively, on PND 180. AMM treated dam weights were not affected, however, there was a 61% decrease in maternal body weight gain in the 100 mg/kg/day atrazine dams during the d osing period. Preputial separation (PPS) was significantly delayed in male offspring by 0.8 days in the 0.87 mg AMM (42.3 d) and 8.73 mg AMM (42.3 d) dose groups, and by 1.6 days in the atrazine group, (43.1 d) compared to controls (41.5 d), however, thes e marginal delays were not considered to be adverse since they were within ±2 day variation. PPS had not occurred by PND44 (a day randomly chosen based on the general window for PPS timing) in 4.5% of control males, ≥13% in the AMM treated groups, and 28% in the ATR treated group. All animals achieved PPS by PND51. Prolactin measures were not affected at PND 120 or 180 (no statistical significance). Serum testosterone levels were increased 60% in the 100 mg ATR group at PND 120 but decreased 60% in the 8.73 mg AMM (60%) at PND 180. Serum estradiol was decreased 33

- 41% at all dosed AMM groups and 40% in the 100 mg ATR, compared to controls. In 100 mg ATR, serum estrone was increased 46% at PND 120 but decreased 47% at PND 180. Despite the different hormone ch anges seen, all mean hormone concentrations were within the normal reported ranges.

At PND 120, anterior pituitary weight was significantly greater in 0.87 AMM (16%) and 100 mg ATR (17%) males compared to controls. The mean ventral prostate weights of 8. 73 mg AMM were statistically decreased (13%) compared to controls at PND 120. No other reproductive tissue weights were affected at PND 120 or PND 180.

The study authors reported a significant increase in the percentage of males exhibiting lipomatous epid idymal fat masses collected at necropsy at PND 120 and 180 at all dose levels. There was also a significant increase in the incidence of pale prostatic foci of discoloration noted in the males at necropsy at both 120 and 180 days at all dose levels.

There was a significant increase in the incidence of prostatitis and in the severity of prostate inflammation in all AMM dose groups (67

- 80%) and the atrazine dose group (81%) on PND 120 compared to controls (48%) (Table 3). The severity scores of affected mal es on PND 120 were greater in all AMM treatment groups than in the ATR exposed group. The incidence of prostatitis and severity scores decreased over time in all AMM dose groups and 100 mg ATR dose group and was diminished by PND 180 (Table 4).Page 205 of 210 There are a number issues with this study which require further discussion.

1. Study design (applicable to both Enoch and Stanko): When conducting a mixture study, one of the critical components is the ability to distinguish the effects of an individual chemical component to the effects of the mixture. Most often, investigators begin with a hypothesis of dose

- additivity or response

- additivity and subsequently test their hypothesis. No such hypothesis testing was performed here. Furthermore, the investigators failed to evaluate each of the individual components in separate dose

response evaluations. There are known differences in potency for atrazine, DACT, DIA, and DEA for some endpoints. As such, with the exception of atrazine, one can not rule out that the findings reported are simply caused by a single chemical.Page 206 of 210 In the specific case of atrazine, pharmacokinetic studies show that the parent compound is rapidly metabolized to DACT, DEA, and DIA. As such, atrazine exposure results in a mixture of metabolites in the body. It would be helpful to have internal dosimetry data for the mixture study to compare the atrazine exposed animals to the AMM exposed animals. The authors have indicated to the Agency that such tissue dose data may be available in the future.

Qualitatively the major difference between the atrazine exposed animals and the AMM exposed animals is the inclusion of the hydroxy metabolite, hydroxyatrazine (HA), in the AMM. HA is not a mammalian metabolite, but is instead an environmental degradate. HA is believed to have a different target organ (kidney) than atrazine, DACT, DEA, and DIA. As such, the investiga tors have potentially developed a mixture using chemicals with different modes of action, further highlighting the importance of the individual component dose

- response curves.
2. Reporting of the findings: In the methods section of the Enoch et al stu dy, the authors state they analyzed all data by dam and block. As such, with n ≥ 6/treatment/block the n for the statistical analysis should be n ≥ 12. The analyses in the figures have n values that often exclude 12. As such, it is difficult to ascertai n if they looked at every endpoint/block or just used the two blocks for different endpoints. In addition, they report that they started with n dams ≥ 6 then sacrificed n ≥5 (2 pups/dam) at PND 4, 25, 33, 40, and 60. It is unclear what happened to the mi ssing dam and respective pups. Lastly, the text below some tables/figures (e.g., Table 2, Figure 4) suggests that statistics were actually done on the pups, not the dams.

Similarly, in the Stanko et al study, unless otherwise indicated, treated dams w ere utilized as the unit of measure for endpoints measured prior to PPS. Although not specifically stated, it follows that after PPS, statistics were done on the pups. It was difficult to track the number of pups in this study. For example, the data pre sented in Figure 4 for Day of PPS shows a potential pup loss in two groups when looking at the n’s in each group (pups n = 67, 47 , 64, 66 and 40 , for 0, 0.09 mg/kg AMM, 0.87 mg/kg AMM, 8.73 mg/kg AMM, and 100 mg/kg atrazine respectively). The study author did not report any sort of litter difference or pup loss to explain this decrease.

3. Subjective scale used to evaluate the mammary gland whole mounts: The investigators use a subjective scale

--- a 1

- 4 scoring system (1 = poor development/structure; 4 = normal development/structure). The scoring criteria varied depending on the age of the pups. This score attempts to capture a number of different characteristics in a single value. As shown in the table above, the reported mean values for all groups, including controls, lie between 1.70 and 3.83 with control means as low as 2.50. As such, the 1

- 4 scale has very little resolution. Furthermore, these values provide no information around the specific characteristics (e.g., branching, budding patterns, e tc) which have been effected. It is also of concern that the control means have been scored as low as 2.50 when a score of 4 is expected to represent normal development/structure.Page 207 of 210 Conclusion: The mixture study, as currently reported, is not sufficientl y robust for use in setting regulatory endpoints. Overall, in both studies, either the dose

- response relationships are weak and/or there are critical concerns with study design and data reporting/analysis, which are discussed above, which preclude meaningf ul conclusions.

A.11 OTHER

A.11.1 Fakhouri, WD, Nunez, JL, Trail, F. (2010?) Atrazine Binds to the Growth Hormone Releasing Hormone Receptor and Affects Growth Hormone Gene Expression. In press

This study concentrated on identifying a ligand bind ing receptor for atrazine in pituitary cells that may explain the mechanism of action at the gene expression level.

The effect of atrazine (ATR) on animals has been difficult to elucidate. Studies on whole animals have correlated low environmental conc entrations (0.1

- 1.0 ppb) with alterations in sexual development in Xenopus laevis along with mammary tumors in rats. Other confounding studies have shown that ‘environmentally relevant’ doses of ATR had little effect on these same subjects. This debate p rompted research to identify possible mechanism of endocrine interference. Specifically, examination of the binding specificity of ATR to the growth hormone releasing hormone receptor (GHRHR) and the effects on the expression of its target gene, growth ho rmone (GH), and the expression of other pituitary hormonal balance genes.

The pituitary cell culture and treatments were thoroughly explained and described, use of PND 7 male rats from breeder females were approved and followed NIH guidelines. Pituitary glands were dissected and cells were dissociated by trituration, with cell number and viability determined by trypan blue exclusion methods. Cultured cells were treated with one of four exposure compounds: ATR, dexamethasone (Dex), RU38486 and/or rat GHR F to determine the competitive inhibition of each compound on the gene expression. All treatments were initiated on day in vitro (DIV) 0. For the combinations of treatments the first agent was administered 30 minutes prior to the application of the

secon d agent. All chemicals were dissolved in DMSO (final DMSO concentration < 0.5% v/v). For real time quantitative PCR (RTqPCR) and competitive inhibition assays, cells were harvested and rinsed with 0.1 M PBS, then covered with RIPA buffer. The trypan blu e exclusion method was used to determine cell number and cells were counted with a hemocytometer. Total RNA was extracted from treated and untreated pituitary cells. For each experiment, results from the treatment replicates were averaged and the amount of mRNA of each gene was normalized to the mRNA of the histone H3 gene. The standard error of each treatment was calculated based on the variation among the replicates of each tested gene, normalized to the standard error of histone H3 in those samples. Each biological experiment was repeated at least two times.

Protein levels in atrazine

- treated pituitary cell (GH, LH, PRL, and CHRH receptor) cultures were determined in AtT

- 20 mouse pituitary tumor cell line (ATCC, Val). InPage 208 of 210 another approach to block a drenocorticotrophic (ACTH) and pituitary adenylate cyclase

activating protein (PACAP) receptors, protein levels of GH in the rat GH3 pituitary tumor cell line were quantified after treatment with 5 and 10 μl ACTHR antibodies at a concentration of 0.2 mg/ml , 15 minutes prior to treatment with 1ppm ATR. This experiment was repeated twice. Competitive binding assays of ATR to pituitary cells were assayed after 0.5, 1.0, 2.0, and 3.0 hours at 37 degrees Celsius. The radioactivity of each vial was measured us ing a scintillation counter. Saturation was measured over a 3 hour time course. No significant increase in binding was observed after 0.5 hours of incubation. Testing the ability of GHRF to competitively inhibit the binding of radio labeled ATR was also performed. Actin and tubulin were immunofluorescently labeled and cells were visualized and imaging was performed.

The results presented describe a remarkable reduction in the mRNA levels of genes encoding GH (growth hormone) and LH (luteinizing hormone ) were found after a 1.0ppm ATR exposure to pituitary cells, while there was an increase mRNA level of PRL (prolactin) in treated cells compared to controls. Expression of GH was reduced in ATR treated cells compared to controls; however, GH expression d id not appear to be inhibited in cells treated with ATR and GHRF

– suggesting that GHRF may displace ATR binding to the GHRHR. Contrary results showed that GH expression was also lower following 1ppm GHRF treatment versus 0.5ppm GHRF. Further, the expres sion was also slightly reduced in cells treated with ATR and 2ppm GHRF compared to cells treated with ATR and 1ppm GHRF. Slight decreases and increases were found in GH, LH and PRL proteins, respectively. Competitive binding was found with ATR only and AT R and GHRF increasing mixtures. The author reported Dex+ATR were comparable to ATR alone and that there didn’t seem to be a remarkable difference when compared to RU38486 alone due to variation within technical replicates. Cells were considered to have b een functioning physiologically when observing histone H3 expression after ATR treatment. Blocking ACTH receptor with their antibodies did not interfere with the antagonistic effect of ATR on GH. Moderate alleviation of the antagonistic effect of ATR

on GH protein levels was observed.

The authors concluded that the ligand

- binding receptor, AGHRHR, was responsive to atrazine. They further conclude that their hypothesis of the pituitary gland being the target of ATR is supported by a Stoker and Cooper, 2007 study which round “a small

co ncentration of ATR present in the anterior pituitary). They observed reductions in the GH and LH expression in cultured rat pituitary cells following ATR exposure. They further insist that LH expression may have been reduced due to a reduction in GH expr ession, resulting from ATR mediated effects on GHRHR. They also attribute mammary tumor formation to an increase in PRL expression in ATR treated pituitary cells. They expand to say that there is an increased somatostain receptor expression which may be secondary to an antagonistic effect of ATR on GHRHR. Their results did lead them to believe that the relatively moderate effect on GH expression in response to ATR on the PACAP receptor shows that ATR is not a primary ligand for the PACAPR.

The authors conclude that ATR targets the GHRHR of pituitary cells and that inhibition of GH production in these cells results from competition between ATR and GHRF forPage 209 of 210 GHRHR binding. They make claims that this is the first time a receptor for ATR has been identifie d. Ultimately, the authors would like us to believe that identification of GHRHR as a target of ATR may facilitate future studies on the effects of ATR and will inform efforts to develop guidelines for safe levels of ATR in water systems.

The Agency revi ewer concludes that the results shown are speculatory at best and the slight or very slight increase and/or decreases seen in competitive binding and protein expression are not definitive.

A.11.2 Yi, KD, Perez, EJ, Breckenridge, CB, and Simpkins, JW (201 0) Ex vivo effects of atrazines or metabolites on phosphodiesterase activity. In press

This study concentrated on phosphodiesterase (PDE) activity and whether atrazine and its metabolites inhibit this process, while also noting effects on aromatase in viv o . It provides several theories on the possible MOA regarding the incidence of mammary tumors, delayed onset of puberty, and immune suppression.

The premise that there is an alternative MOA for mammary tumors in female Sprague

Dawley rats is that mRNA ex pression and aromatase activity was increased in H295R adrenocortical carcinoma cells when exposed to a minimum of 10μM atrazine. Increased cyclic AMP (cAMP) levels were correlated with the induction of aromatase activity by atrazine; increases in cAMP ma y be mediated either via increases in adenylyl cyclase which catalyzes the conversion of ATP to cAMP, or via the inhibition of PDE which catalyzes the breakdown of cAMP to AMP. Therefore, high concentrations of atrazine in vitro may be mediated by increas es in intracellular cAMP, activating adenylyl cyclase, or by inhibiting PDE.

Atrazine, deethyl atrazine (DEA), deisopropyl atrazine (DIA), diaminochlorotriazine (DACT), ammeline (AM), and hydroxyatrazine (hydroxyl

- ATZ) used were approximately 97% pure. IBMX, 3

- isobutyl

- 1

- methylxanthine was used as the positive control. The IMAP phosphodiesterase assay kit was used along with bovine PDE at varying concentrations (0.000025 to 0.5 PDE U/ml in 96

- well plates). To determine the effects of addition of atraz ine after completion of the PDE reaction, an hour long incubation period was allowed and then to assess the effects of atrazine and its metabolites on PDE activity, the potential inhibitors were added at concentrations ranging from 5pM to 5 nM in 10 μl of assay buffer enzyme with additional 90 minute substrate/incubation period. Competitive inhibition studies were similarly conducted with the addition of varying concentrations of DACT with atrazine. Determining IC 50 values was done using a 4

- parameter log curve and creating sigmoidal standard curves.

The results from the optimized assay for PDE inhibition showed a concentration

dependent increase in the relative fluorescence output of the assay at both 30 minutes and 2 hours after the addition of the bi nding reagent. Maximal output, of 0.025 PDE U/ml was used for all subsequent assays. Addition of atrazine after completion of the enzymatic reaction didn’t have any effect on the fluorescence output of the assay.Page 210 of 210 Regarding the effects of atrazine and to a much lesser extent its metabolites inhibited PDE in a cell

- free system. The IC 50 for atrazine (39.52 μM) while for IBMX it was 1.749 μM. The IC 50 for DIA was 1371 μM and DEA at 7156 μM were 35 and 181

- fold less potent inhibitors of PDE than was atrazi ne; all other metabolites were inactive. DACT, however dose

- dependently caused a rightward shift in the dose

- response curve of the atrazine mediated inhibition of PDE activity suggesting to the authors that although DACT does not inhibit PDE out right, it perhaps modifies the effect of atrazine on PDE. Also at both 5 and 50 μM concentrations of DACT, the IC 50 of atrazine was several folds higher, suggesting competitive inhibitory effects of DACT.

The authors concluded that atrazine at all concentrations which exceed its aqueous solubility limits, is a weak inhibitor of PDE and that its metabolites have extremely weak, or no effect on PDE activity. The primary metabolite of atrazine, DACT was shown to act as a competitive inhibitor of atrazine

- induced inh ibition of PDE activity. Although, not supported, the authors elude to the possibility that the inhibition by atrazine of PDE in ex vivo condition, a cell

- free system with a complex mixture of different PDE isozymes and substrate, could be due to a non

- specific effect of the precipitate on the kinetics of the reaction. The IC 50 for atrazine

- mediated inhibition of PDE activity in this stud y (40 μM) was vastly different, 22

- fold higher, form the concentration reported by Roberge et al (2004). No true rationale was given for this discrepancy

– however, they went on to describe the many similarities in results from the two laboratories. Even at high concentrations of atrazine there have been no shown increases in aromatase mRNA or enzyme activity in vivo with the exception of a study on juvenile zebrafish. Therefore the study concludes that a general or local increase in the expression of t his enzyme in vivo , would not account for the observed effects of high doses of atrazine on estrous cycles, mammary tumors or the age of onset of puberty.

In mammals, atrazine is NOT acutely toxic (oral LD= 3090 mg/kg) and is only moderately toxic follo wing repeat dosing to rats, mice, rabbits, or dogs. Atrazine

mediated inhibition of PDE ex vivo is unlikely to account for the tissue, sex, strain and species

- specific effects of chronic high

- dose exposure to atrazine. The bottom

- line is that it is unlik ely that an atrazine

- mediated inhibition PDE activity would occur at environmentally relevant concentrations. The potential human exposure to atrazine is low and equally unlikely to result in PDE inhibition in humans. The study goes on to endorse the NOE L (1.8 mg/kg/day) which is currently used to regulate chronic exposure to atrazine and its chlorometabolites and appears to be protective of any potential effects in humans.